Neuroprotection using nap-like and sal-like peptide mimetics

ABSTRACT

This invention relates to NAP-like and SAL-like peptide mimetics, polypeptides, or small molecules derived from them, and their use in the treatment of neuronal dysfunction, neurodegenerative disorders cognitive deficits, neuropsychiatric disorders, and autoimmune disease.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/708,384, filed Feb. 18, 2010, which is a continuation-in-part ofPCT/CA2008/001497, filed Aug. 22, 2008, which claims the benefit of U.S.Provisional Application No. 60/957,790, filed Aug. 24, 2007, thecontents of all of the above are hereby incorporated by reference in theentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“SEQTXT_(—)85085-854881_(—)000820US.txt” created Oct. 30, 2012 andcontaining 7,101 bytes. The material contained in this text file isincorporated by reference.

FIELD OF THE INVENTION

This invention relates to NAP-like and SAL-like peptide mimetics,polypeptides, or small molecules derived from them, and their use in thetreatment of neuronal dysfunction, neurodegenerative disorders cognitivedeficits, neuropsychiatric disorders, and autoimmune disease.

BACKGROUND OF THE INVENTION

NAP, an 8-amino-acid peptide (NAPVSIPQ, SEQ ID NO:1), is derived fromactivity-dependent neuroprotective protein, ADNP (U.S. Pat. No.6,613,740; Bassan et al., J. Neurochem. 72: 1283-1293 (1999)). The NAPsequence within the ADNP gene is identical in rodents and humans (U.S.Pat. No. 6,613,740; Zamostiano, et al., J. Biol. Chem. 276:708-714(2001)).

In cell cultures, NAP has been shown to have neuroprotective activity atfemtomolar concentrations against a wide variety of toxins (Bassan etal., 1999; Offen et al., Brain Res. 854:257-262 (2000)). In animalmodels simulating parts of the Alzheimer's disease pathology, NAP wasprotective as well (Bassan et al., 1999; Gozes et al., J. Pharmacol.Exp. Ther. 293:1091-1098 (2000); see also U.S. Pat. No. 6,613,740). Innormal aging rats, intranasal administration of NAP improved performancein the Morris water maze. (Gozes et al., J. Mol. Neurosci. 19:175-178(2002)). Furthermore, NAP reduced infarct volume and motor functiondeficits after ischemic injury, by decreasing apoptosis (Leker et al.,Stroke 33:1085-1092 (2002)) and reducing damage caused by closed headinjury in mice by decreasing inflammation (Beni Adani et al., J.Pharmacol. Exp. Ther. 296:57-63 (2001); Romano et al., J. Mol. Neurosci.18:37-45 (2002); Zaltzman et al., NeuroReport 14:481-484 (2003)). In amodel of fetal alcohol syndrome, fetal death after intraperitonealinjection of alcohol was inhibited by NAP treatment (Spong et al., J.Pharmacol. Exp. Ther. 297:774-779 (2001); see also International PCTApplication Publication No. WO 00/53217). Utilizing radiolabeledpeptides these studies showed that NAP can cross the blood-brain barrierand can be detected in rodents' brains either after intranasal treatment(Gozes et al., 2000) or intravenous injection (Leker et al., 2002) orintraperitoneal administration (Spong et al., 2001).

SAL, a 9-amino acid peptide (SALLRSIPA, SEQ ID NO:19), also known asADNF-9 or ADNF-1, was identified as the shortest active form of ADNF(see U.S. Pat. No. 6,174,862). SAL has been shown in in vitro assays andin vivo disease models to keep neurons of the central nervous systemalive in response to various insults (e.g., Gozes et al., 2000;Brenneman et al., J. Pharmacol. Exp. Ther. 285:619-627 (1998)). D-SAL isan all D-amino acid derivative of SAL that is stable and orallyavailable (Brenneman, et al., J. Pharmacol Exp Ther. 309:1190-7 (2004))and surprisingly exhibits similar biological activity (potency andefficacy) to SAL in the systems tested. ADNF-1 complexes are describedin International PCT Application Publication No. WO03/022226.

Neuroactive peptides, such as NAP and SAL, appear to be extremelysensitive to even single-amino acid, conservative substitutions. See,e.g., Brenneman et al., J. Pharm. Ex. Ther., 285:619-627 (1998) andWilkemeyer et al., Proc. Natl. Acad. Sci, USA, 100:8543-8 (2003). Thus,while NAP and SAL are model neuroactive peptides, even conservativepeptide variations of their core sequences are not predicted to betherapeutically effective. Accordingly, while there have been advancesin this field, there remains a need for further neuroactive peptides.The present invention solves this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides biologically active NAP-like peptidemimetics or SAL-like pepetide mimetics and methods to make and use thesepeptides. The formula of the NAP-like peptide mimetics or SAL-likepepetide mimetics is (R¹)_(a)—(R²)—(R³)_(b). R¹ is an amino acidsequence comprising from 1 to about 40 amino acids wherein each aminoacid is independently selected from the group consisting of naturallyoccurring amino acids and amino acid analogs. R² is one of the followingsequences: NATLSIHQ (SEQ ID NO:4), STPTAIPQ (SEQ ID NO:6), NAVLSIHQ (SEQID NO:2), NATLSVHQ (SEQ ID NO:3), NATLSIVHQ (SEQ ID NO:5), NTPVSIPQ (SEQID NO:7), APVSIPQ (SEQ ID NO:8), NTPISIPQ (SEQ ID NO:9), NAPVSIP (SEQ IDNO:10), NAPVAVPQ (SEQ ID NO:11), NARVSIPQ (SEQ ID NO:12), DAPVSVPQ (SEQID NO:13), ALLRSIPA (SEQ ID NO:20), ALLRSIP (SEQ ID NO:21), AMLRSIPA(SEQ ID NO:22), ALLRAIPA (SEQ ID NO:23), SALLRSIP (SEQ ID NO:24),SALLRAIP (SEQ ID NO:25), ALLRTIPA (SEQ ID NO:26), and ALLRSVPA (SEQ IDNO:27). R³ is an amino acid sequence comprising from 1 to about 40independently selected amino acids, e.g., naturally occurring aminoacids or amino acid analogs. a and b are independently selected and areequal to zero or one. The sequences NAPVSIPQ (SEQ ID NO:1) or SALLRSIPA(SEQ ID NO:19) are specifically excluded from this formula.

In one embodiment, the NAP-like peptide mimetic or SAL-like peptidemimetic includes a core sequence, i.e., R² selected from NATLSIHQ (SEQID NO:4) and STPTAIPQ (SEQ ID NO:6).

In another embodiment, the NAP-like peptide mimetic or SAL-like peptideincludes only the core amino acid sequence, i.e., R². That is, a and bare equal to zero.

In one embodiment, the NAP-like peptide mimetic or SAL-like peptideincludes at least one D-amino acid in the core amino acid sequence,i.e., R².

In one embodiment, each amino acid of the NAP-like peptide mimetic orSAL-like peptide, i.e., R², is a D-amino acid.

In another embodiment, the NAP-like peptide mimetic or SAL-like peptidemimetic includes at least one protecting group.

In one embodiment, the NAP-like peptide mimetic or SAL-like peptidemimetic includes the core amino acid sequence NATLSIHQ (SEQ ID NO:4). Ina further embodiment, the NAP-like peptide mimetic or SAL-like peptidemimetic consists of the core amino acid sequence NATLSIHQ (SEQ ID NO:4).In a further embodiment, the core amino acid sequence NATLSIHQ (SEQ IDNO:4) includes at least one D-amino acid. In another embodiment, eachamino acid of the core amino acid sequence NATLSIHQ (SEQ ID NO:4) is aD-amino acid.

In one embodiment, the NAP-like peptide mimetic or SAL-like peptidemimetic includes the core amino acid sequence STPTAIPQ (SEQ ID NO:6). Ina further embodiment, the NAP-like peptide mimetic or SAL-like peptidemimetic consists of the core amino acid sequence STPTAIPQ (SEQ ID NO:6).In a further embodiment, the core amino acid sequence STPTAIPQ (SEQ IDNO:6) includes at least one D-amino acid. In another embodiment, eachamino acid of the core amino acid sequence STPTAIPQ (SEQ ID NO:6) is aD-amino acid.

In another aspect, the invention provides a pharmaceutical compositionincludes a NAP-like peptide mimetic or SAL-like peptide mimetic with theformula described above. The pharmaceutical composition can also includea second neuroprotective polypeptide such as a neuroprotectivepolypeptide comprising NAPVSIPQ (SEQ ID NO:1) or SALLRSIPA (SEQ IDNO:19).

In another aspect the invention provides a method of treating orpreventing a neurodegenerative disorder, a cognitive deficit, anautoimmune disorder, peripheral neurotoxicity, motor dysfunction,sensory dysfunction, anxiety, depression, schizophrenia, psychosis, acondition related to fetal alcohol syndrome, a condition involvingretinal degeneration, a disorder affecting learning and memory, or aneuropsychiatric disorder in a subject, by administering atherapeutically effective amount of a NAP-like peptide mimetic orSAL-like peptide mimetic with the formula listed above, to a subject inneed of treatment, thereby treating or preventing the neurodegenerativedisorder, the cognitive deficit, the autoimmune disorder, peripheralneurotoxicity, motor dysfunction, sensory dysfunction, anxiety,depression, schizophrenia, psychosis, the condition related to fetalalcohol syndrome, the condition involving retinal degeneration, thedisorder affecting learning and memory, or the neuropsychiatric disorderin the subject. In a preferred embodiment, the administered NAP-likepeptide mimetic or SAL-like peptide mimetic includes one of thefollowing amino acid sequences: NATLSIHQ (SEQ ID NO:4) and STPTAIPQ (SEQID NO:6).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The effect of peptides on survival of astrocytes followingincubation with 200 mM ZnCl₂ for 4 hrs. The graph depicts at least 3experiments per peptide which were each performed in quintuplets.NATLSIHQ (SEQ ID NO:4): *=p<0.05; **=p<0.005, ***=p<0.0005; STPTAIPQ(SEQ ID NO:6): #=p<0.05 (In comparison to the negative control—noadditions).

FIG. 2: The effect of peptides on the survival of neuroglial culturesfollowing intoxication with beta-amyloid. The graph depicts 3experiments per peptide which were each performed in quintuplets.NATLSIHQ (SEQ ID NO:4): *=p<0.05; **=p<0.005; STPTAIPQ (SEQ ID NO:6):#=p<0.05. (In comparison to the negative control—no additions).

FIG. 3: Time course of VQIVYK aggregation at different concentrations(1-500 μM) in the presence of 250 μM polyglutamate and 20 μM MOPS. Peakaggregation occurs on day 7 with 100 μM VQIVYK in 250 μM polyglutamateand 20 μM MOPS at pH 6.5.

FIG. 4: Effect of peptides NAPVSIPQ and NATLSIHQ on VQIVYK aggregation.NATLSIHQ (SEQ ID NO:4) shows superior effect than NAPVSIPQ (SEQ ID NO:1)in the inhibition of tau aggregation. Key: ###=p<0.0005 NAP vs. control;***=p<0.0005 NATLSIHQ vs. control; and **=p<0.001 NATLSIHQ vs. control.The graph represents three independent experiments performed inquadruplicates.

FIG. 5: The Morris water maze results on Day 5 between the non-Tg miceand the Tau-tg vehicle treated mice show a statistically marginallysignificant difference.

FIG. 6: Latency to find the hidden platform during the second dailytrial. The improvement in learning was analyzed using t-tests fordependent samples that compared for each group the latency to find theplatform on the first day and on the fifth day of the MWM. Significantimprovement was found in the Tau-Tg NAT treated group (p=0.039) and forthe non-Tg group (p=0.007).

FIG. 7: Brain-Body weight ratios show protective effect of NAT treatmentfrom neurodegeneration. Brain-Body weight ratio was calculated for eachmouse and averaged per group. [TAU-Tg+NAT 0.0148+0.0009, TAU-Tg+Vh0.0117+0.0007, w.t 0.0152+0.0005]. * Tukey HSD post-hoc test showed asignificant difference between the NAT and vehicle treated TAU-Tg groups(p=0.030). ** Tukey HSD post-hoc test showed a significant differencebetween the non-Tg and the vehicle treated Tau-tg animals (p=0.007).

FIG. 8: NAT treatment leads to a statistically significant increase inthe amount of ADNP protein in cell nucleus. The ADNP amount in each bandwas calculated as percentage from the total amount of all bands. ADNPamounts of each group were averaged-TAU-Tg+NAT 10.747+0.859, TAU-Tg+Vh5.52+0.92, w.t 11.17+1.35]. * Tukey HSD post-hoc test revealed adifference between the NAT and vehicle treated TAU-Tg groups (p=0.0028).** Tukey HSD post-hoc test revealed a difference between the vehicletreated TAU-Tg and non-TG group (p=0.0097). Vh=vehicle.

FIG. 9: NAT treatment leads to no significant change in the amount ofactin in cell nucleus. The actin amount in each band was calculated asits percentage from the total amount of all bands. Actin amounts of eachgroup were averaged-TAU-Tg+NAT 10.68+1.746, TAU-Tg+Vh 8.404+3.4, non-Tg8.295+2.61.

DEFINITIONS

The phrases “NAP-like peptide mimetics” and “NAP-like peptides” referequally to both peptides and mimetics that have similarity to NAP(NAPVSIPQ) (SEQ ID NO:1). The phrases therefore refer to peptides andmimetics comprising a sequence having the following formula:(R¹)_(a)—(R²)— (R³)_(b), where R¹ and R³ are independently selected andare amino acid sequences comprising from 1 to about 40 amino acidswherein each amino acid is independently selected from the groupconsisting of naturally occurring amino acids and amino acid analogs; R²is a NAP-like peptide such as: NAVLSIHQ (SEQ ID NO:2), NATLSVHQ (SEQ IDNO:3), NATLSIHQ (SEQ ID NO:4), NATLSIVHQ (SEQ ID NO:5), STPTAIPQ (SEQ IDNO:6), NTPVSIPQ (SEQ ID NO:7), APVSIPQ (SEQ ID NO:8), NTPISIPQ (SEQ IDNO:9), NAPVSIP (SEQ ID NO:10), NAPVAVPQ (SEQ ID NO:11), NARVSIPQ (SEQ IDNO:12), DAPVSVPQ (SEQ ID NO:13), NXPVSIPQ (SEQ ID NO:14), NXP+SIPQ (SEQID NO:15), NAPV++PQ (SEQ ID NO:16), NAXVSIPQ (SEQ ID NO:17) and +APVS+PQ(SEQ ID NO:18), wherein X refers to any amino acid and +refers to aconservative amino acid; and a and b are independently selected and areequal to zero or one, with the proviso that the NAP-like peptide mimeticis not NAP. The phrase also refers to D-amino acid analogs, for examplewhere as few as one or as many as all amino acids are in the Dconfiguration.

The phrases “SAL-like peptide mimetics” and “SAL-like peptides” referequally to both peptides and mimetics that have similarity to SAL(SALLRSIPA) (SEQ ID NO:19). The phrases therefore refer to peptidescomprising a sequence having the following formula: (R¹)_(a)—(R²)—(R³)_(b), where R¹ and R³ are independently selected and are amino acidsequences comprising from 1 to about 40 amino acids wherein each aminoacid is independently selected from the group consisting of naturallyoccurring amino acids and amino acid analogs; R² is a SAL-like peptidesuch as: ALLRSIPA (SEQ ID NO:20), ALLRSIP (SEQ ID NO:21), AMLRSIPA (SEQID NO:22), ALLRAIPA (SEQ ID NO:23), SALLRSIP (SEQ ID NO:24), SALLRAIP(SEQ ID NO:25), ALLRTIPA (SEQ ID NO:26), ALLRSVPA (SEQ ID NO:27),A+LRSIPA (SEQ ID NO:28), ALLR+IPA (SEQ ID NO:29), SALLR+IP (SEQ IDNO:30), and ALLRS+PA (SEQ ID NO:31) wherein X refers to any amino acidand +refers to a conservative amino acid; and a and b are independentlyselected and are equal to zero or one, with the proviso that theSAL-like peptide mimetic is not SAL. The phrase also refers to D-aminoacid analogs, for example where as few as one or as many as all aminoacids are in the D configuration.

The phrase “ADNF polypeptide” refers to one or more activity dependentneurotrophic factors (ADNF) that have an active core site comprising theamino acid sequence of NAPVSIPQ (SEQ ID NO:1) (referred to as “NAP”) orSALLRSIPA (SEQ ID NO:19) (referred to as “SAL”) and that haveneurotrophic/neuroprotective activity as measured with in vitro corticalneuron culture assays described by, e.g., Hill et al., Brain Res.603:222-233 (1993); Brenneman & Gozes, J. Clin. Invest. 97:2299-2307(1996); and Forsythe & Westbrook, J. Physiol. Lond. 396:515 (1988). AnADNF polypeptide can be an ADNF I polypeptide, an ADNF III polypeptide,their alleles, polymorphic variants, analogs, interspecies homolog, anysubsequences thereof (e.g., SALLRSIPA (SEQ ID NO:19) or NAPVSIPQ (SEQ IDNO:1)) or lipophilic variants that exhibit neuroprotective/neurotrophicaction on, e.g., neurons originating in the central nervous systemeither in vitro or in vivo. An “ADNF polypeptide” can also refer to amixture of an ADNF I polypeptide and an ADNF III polypeptide.

The phrase “ADNF III polypeptide” or “ADNF III,” also calledactivity-dependent neuroprotective protein (ADNP), refers to one or moreactivity dependent neurotrophic factors (ADNF) that have an active coresite comprising the amino acid sequence of NAPVSIPQ (SEQ ID NO:1)(referred to as “NAP”) and that have neurotrophic/neuroprotectiveactivity as measured with in vitro cortical neuron culture assaysdescribed by, e.g., Hill et al., Brain Res. 603, 222-233 (1993); andGozes et al., Proc. Natl. Acad. Sci. USA 93, 427-432 (1996). An ADNFpolypeptide can be an ADNF III polypeptide, allelelic or polymorphicvariant, analog, interspecies homolog, or any subsequences thereof(e.g., NAPVSIPQ; SEQ ID NO:1) that exhibit neuroprotective/neurotrophicaction on, e.g., neurons originating in the central nervous systemeither in vitro or in vivo. ADNF III polypeptides can range from abouteight amino acids and can have, e.g., between 8-20, 8-50, 10-100 orabout 1000 or more amino acids.

Full length human ADNF III has a predicted molecular weight of 123,562.8Da (>1000 amino acid residues) and a theoretical pI of about 6.97. Asdescribed above, ADNF III polypeptides have an active site comprising anamino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:1)(also referred to as “NAPVSIPQ” or “NAP”). See Zamostiano et al., J.Biol. Chem. 276:708-714 (2001) and Bassan et al., J. Neurochem.72:1283-1293 (1999). Unless indicated as otherwise, “NAP” refers to apeptide having an amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln(SEQ ID NO:1), not a peptide having an amino acid sequence ofAsn-Ala-Pro. Full-length amino acid and nucleic acid sequences of ADNFIII can be found in International PCT Application Publication Nos. WO98/35042, WO 00/27875, U.S. Pat. Nos. 6,613,740 and 6,649,411. TheAccession number for the human sequence is NP_(—)852107, see alsoZamostiano et al., supra.

The term “ADNF I” refers to an activity dependent neurotrophic factorpolypeptide having a molecular weight of about 14,000 Daltons with a pIof 8.3±0.25. As described above, ADNF I polypeptides have an active sitecomprising an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala(SEQ ID NO:19) (also referred to as “SALLRSIPA” or “SAL” or “ADNF-9”).See Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996), Glazner etal., Anat. Embryol. ((Berl). 200:65-71 (1999), Brenneman et al., J.Pharm. Exp. Ther., 285:619-27 (1998), Gozes & Brenneman, J. Mol.Neurosci. 7:235-244 (1996), and Gozes et al., Dev. Brain Res. 99:167-175(1997). Unless indicated as otherwise, “SAL” refers to a peptide havingan amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ IDNO:19), not a peptide having an amino acid sequence of Ser-Ala-Leu. Afull length amino acid sequence of ADNF I can be found in InternationalPCT Application Publication No. WO 96/11948.

The term “subject” refers to any mammal, in particular human, at anystage of life.

The term “contacting” is used herein interchangeably with the following:combined with, added to, mixed with, passed over, incubated with, flowedover, etc. Moreover, the polypeptides or nucleic acids of the presentinvention can be “administered” by any conventional method such as, forexample, parenteral, oral, topical, nasal, and inhalation routes. Insome embodiments, parenteral and nasal or inhalation routes areemployed.

The term “biologically active” refers to a peptide sequence that willinteract with naturally occurring biological molecules to eitheractivate or inhibit the function of those molecules in vitro or in vivo.The term “biologically active” is most commonly used herein to refer toNAP-like peptide mimetics that exhibit neuroprotective/neurotrophicaction on neurons originating in the central nervous system both invitro or in vivo. Thus, the present invention provides polypeptidesubsequences that have the same or similar activity as NAP when tested,e.g., cerebral cortical cultures treated with a neurotoxin (see Gozes etal. Proc. Nat'l. Acad. Sci. USA 93:427-432 (1996)). The peptides canalso be tested as described herein to determine their ability to competewith NAP-tubulin binding by at least 2-10%, preferably greater than 10%.

The phrase “neurodegenerative disorders or cognitive deficits” includes,but is not limited to the following conditions: diseases of centralmotor systems including degenerative conditions affecting the basalganglia (Huntington's disease, Wilson's disease, striatonigraldegeneration, corticobasal ganglionic degeneration), Tourette'ssyndrome, Parkinson's disease, progressive supranuclear palsy,progressive bulbar palsy, familial spastic paraplegia, spinomuscularatrophy, ALS and variants thereof, dentatorubral atrophy,olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration,and dopamine toxicity; diseases affecting sensory neurons such asFriedreich's ataxia, diabetes, peripheral neuropathy, and retinalneuronal degeneration; diseases of limbic and cortical systems such ascerebral amyloidosis, Pick's atrophy, and Retts syndrome;neurodegenerative pathologies involving multiple neuronal systems and/orbrainstem including Alzheimer's disease, Parkinson's disease,AIDS-related dementia, Leigh's disease, diffuse Lewy body disease,epilepsy, multiple system atrophy, Guillain-Barre syndrome, lysosomalstorage disorders such as lipofuscinosis, late-degenerative stages ofDown's syndrome, Alper's disease, vertigo as result of CNS degeneration,ALS, corticobasal degeneration, and progressive supranuclear palsy;pathologies associated with developmental retardation and learningimpairments, Down's syndrome, and oxidative stress induced neuronaldeath; pathologies arising with aging and chronic alcohol or drug abuseincluding, for example, (i) with alcoholism, the degeneration of neuronsin locus coeruleus, cerebellum, cholinergic basal forebrain, (ii) withaging, degeneration of cerebellar neurons and cortical neurons leadingto cognitive and motor impairments, and (iii) with chronic amphetamineabuse, degeneration of basal ganglia neurons leading to motorimpairments; pathological changes resulting from focal trauma such asstroke, focal ischemia, vascular insufficiency, hypoxic-ischemicencephalopathy, hyperglycemia, hypoglycemia, closed head trauma, anddirect trauma; pathologies arising as a negative side-effect oftherapeutic drugs and treatments (e.g., degeneration of cingulate andentorhinal cortex neurons in response to anticonvulsant doses ofantagonists of the NMDA class of glutamate receptor).

“Peripheral neurotoxicity” may be identified and diagnosed in a subjectby a variety of techniques. Typically it may be measured by motordysfunction, muscle wasting, or a change in sense of smell, vision orhearing, or changes in deep tendon reflexes, vibratory sense, cutaneoussensation, gait and balance, muscle strength, orthostatic bloodpressure, and chronic or intermittent pain. In humans these symptoms arealso sometimes demonstrative of toxic effects in both the PNS and theCNS. Ultimately, there are hundreds of possible peripheral neuropathiesthat may result from neurotoxicity. Reflecting the scope of PNSactivity, symptoms may involve sensory, motor, or autonomic functions.They can be classified according to the type of affected nerves and howlong symptoms have been developing. Peripheral neurotoxicity can beinduced by chemotherapeutic agents (anti-cancer, anti-microbial and thelike) and by disease processes. (See, e.g., U.S. patent application Ser.No. 11/388,634).

“Conditions involving retinal degeneration” include, but are not limitedto, laser-induced retinal damage and ophthalmic diseases, such asglaucoma, Retinitis pigmentosa, Usher syndrome, artery or veinocclusion, diabetic retinopathy, retrolental fibroplasias or retinopathyof prematurity (R.L.F./R.O.P.), retinoschisis, lattic degeneration, andmacular degeneration.

A “mental disorder” or “mental illness” or “mental disease” or“psychiatric or neuropsychiatric disease or illness or disorder” refersto mood disorders (e.g., major depression, mania, and bipolardisorders), psychotic disorders (e.g., schizophrenia, schizoaffectivedisorder, schizophreniform disorder, delusional disorder, briefpsychotic disorder, and shared psychotic disorder), personalitydisorders, anxiety disorders (e.g., obsessive-compulsive disorder andattention deficit disorders) as well as other mental disorders such assubstance -related disorders, childhood disorders, dementia, autisticdisorder, adjustment disorder, delirium, multi-infarct dementia, andTourette's disorder as described in Diagnostic and Statistical Manual ofMental Disorders, Fourth Edition, (DSM IV) (see also Benitez-King G. etal., Curr Drug Targets CNS Neurol Disord. 2004 December; 3(6):515-33.Review). Typically, such disorders have a complex genetic and/or abiochemical component.

A “mood disorder” refers to disruption of feeling tone or emotionalstate experienced by an individual for an extensive period of time. Mooddisorders include major depression disorder (i.e., unipolar disorder),mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and manyothers. See, e.g., Diagnostic and Statistical Manual of MentalDisorders, Fourth Edition, (DSM IV).

“Major depression disorder,” “major depressive disorder,” or “unipolardisorder” refers to a mood disorder involving any of the followingsymptoms: persistent sad, anxious, or “empty” mood; feelings ofhopelessness or pessimism; feelings of guilt, worthlessness, orhelplessness; loss of interest or pleasure in hobbies and activitiesthat were once enjoyed, including sex; decreased energy, fatigue, being“slowed down”; difficulty concentrating, remembering, or makingdecisions; insomnia, early-morning awakening, or oversleeping; appetiteand/or weight loss or overeating and weight gain; thoughts of death orsuicide or suicide attempts; restlessness or irritability; or persistentphysical symptoms that do not respond to treatment, such as headaches,digestive disorders, and chronic pain. Various subtypes of depressionare described in, e.g., DSM IV.

“Bipolar disorder” is a mood disorder characterized by alternatingperiods of extreme moods. A person with bipolar disorder experiencescycling of moods that usually swing from being overly elated orirritable (mania) to sad and hopeless (depression) and then back again,with periods of normal mood in between. Diagnosis of bipolar disorder isdescribed in, e.g., DSM IV. Bipolar disorders include bipolar disorder I(mania with or without major depression) and bipolar disorder II(hypomania with major depression), see, e.g., DSM IV.

“Anxiety,” “anxiety disorder,” and “anxiety-related disorder refer topsychiatric syndromes characterized by a subjective sense of unease,dread, or foreboding, e.g., panic disorder, generalized anxietydisorder, attention deficit disorder, attention deficit hyperactivedisorder, obsessive-compulsive disorder, and stress disorders, e.g.,acute and post-traumatic. Diagnostic criteria for these disorders arewell known to those of skill in the art (see, e.g., Harrison'sPrinciples of Internal Medicine, pp. 2486-2490 (Wilson et al., eds.,12th ed. 1991) and DSM IV).

An “autoimmune disorder” refers to an autoimmune disease such asmultiple sclerosis, myasthenia gravis, Guillan-Barre syndrome(antiphospholipid syndrome), systemic lupus erytromatosis, Behcet'ssyndrome, Sjogrens syndrome, rheumatoid arthritis, Hashimoto'sdisease/hypothyroiditis, primary biliary cirrhosis, mixed connectivetissue disease, chronic active hepatitis, Graves'disease/hyperthyroiditis, scleroderma, chronic idiopathicthrombocytopenic purpura, diabetic neuropathy and septic shock (see,e.g., Schneider A. et al., J Biol. Chem. 279:55833-9 (2004)).

“Motor dysfunctions” include muscle wasting and changes in gait,balance, and muscle strength. “Sensory dysfunctions” may be measured bychanges in sense of smell, vision or hearing, or changes in deep tendonreflexes, vibratory sense, cutaneous sensation, or chronic orintermittent pain. Sometimes sensory dysfunctions are associated withdisease, and can be experienced as pain or pins-and-needles, burning,crawling, or prickling sensations, e.g., in the feet and lower legs. Inhumans, both motor and sensory dysfunctions indicate effects in both thePNS and the CNS which may be caused by chemical (e.g.,chemotherapeutics) or disease states.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.Generally, a peptide refers to a short polypeptide. The terms apply toamino acid polymers in which one or more amino acid residue is an analogor mimetic of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. For thepurposes of this application, amino acid analogs refers to compoundsthat have the same basic chemical structure as a naturally occurringamino acid, i.e., an a carbon that is bound to a hydrogen, a carboxylgroup, an amino group, and an R group, e.g., homoserine, norleucine,methionine sulfoxide, methionine methyl sulfonium. Such analogs havemodified R groups (e.g., norleucine) or modified peptide backbones, butretain the same basic chemical structure as a naturally occurring aminoacid. For the purposes of this application, amino acid mimetics refersto chemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may include those having non-naturally occurringD-chirality, as disclosed in International PCT Application PublicationNo. WO 01/12654, which may improve oral availability and other drug likecharacteristics of the compound. In such embodiments, one or more, andpotentially all of the amino acids of NAP-like or SAL-like peptidemimetics will have D-chirality. The therapeutic use of peptides can beenhanced by using D-amino acids to provide longer half life and durationof action. However, many receptors exhibit a strong preference forL-amino acids, but examples of D-peptides have been reported that haveequivalent activity to the naturally occurring L-peptides, for example,pore-forming antibiotic peptides, beta amyloid peptide (no change intoxicity), and endogenous ligands for the CXCR4 receptor. In thisregard, NAP-like or SAL-like peptide mimetics also retain activity inthe D-amino acid form.

Amino acids may be referred to by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Serine (S), Threonine (T);    -   3) Aspartic acid (D), Glutamic acid (E);    -   4) Asparagine (N), Glutamine (Q);    -   5) Cysteine (C), Methionine (M);    -   6) Arginine (R), Lysine (K), Histidine (H);    -   7) Isoleucine (I), Leucine (L), Valine (V); and    -   8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,        Creighton, Proteins (1984)).

One of skill in the art will appreciate that many conservativevariations of the nucleic acid and polypeptide sequences provided hereinyield functionally identical products. For example, due to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions of a nucleic acid sequence that do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence that encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties (see the definitions section), are alsoreadily identified as being highly similar to a disclosed amino acidsequence, or to a disclosed nucleic acid sequence that encodes an aminoacid.

In addition, certain protecting groups may be added to peptidesaccording to the invention. The protecting group may be added to eitherthe N-terminal or C-terminal end of the peptide, or both. As usedherein, the term “protecting group” refers to a compound that renders afunctional group unreactive, but is also removable so as to restore thefunctional group to its original state. Such protecting groups are wellknown to one of ordinary skill in the art and include compounds that aredisclosed in “Protective Groups in Organic Synthesis”, 4th edition, T.W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006. Examplesof protecting groups include, but are not limited to: Fmoc(9-fluorenylmethyl carbamate, Boc, benzyloxy-carbonyl (Z), alloc(allyloxycarbonyl), and lithographic protecting groups.

The terms “isolated,” “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state.

“An amount sufficient” or “an effective amount” or a “therapeuticallyeffective amount” is that amount of a given NAP-like or SAL-like peptidemimetic that exhibits the activity of interest or which provides eithera subjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Intherapeutic applications, the NAP-like or SAL-like peptide mimetics ofthe invention are administered to a patient in an amount sufficient toreduce or eliminate symptoms. An amount adequate to accomplish this isdefined as the “therapeutically effective dose.” The dosing range varieswith the NAP-like or SAL-like peptide mimetic used, the route ofadministration and the potency of the particular NAP-like or SAL-likepeptide mimetic, and the presence or absence of additional therapeuticcompounds in the pharmaceutical composition.

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity, e.g., ligands, agonists, antagonists, andtheir homologs and mimetics. The term “modulator” includes inhibitorsand activators Inhibitors are agents that, e.g., inhibit expression of apolypeptide or polynucleotide of the invention or bind to, partially ortotally block stimulation or enzymatic activity, decrease, prevent,delay activation, inactivate, desensitize, or down regulate the activityof a polypeptide or polynucleotide of the invention, e.g., antagonists.Activators are agents that, e.g., induce or activate the expression of apolypeptide or polynucleotide of the invention or bind to, stimulate,increase, open, activate, facilitate, enhance activation or enzymaticactivity, sensitize or up regulate the activity of a polypeptide orpolynucleotide of the invention, e.g., agonists. Modulators includenaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Assays to identify inhibitors andactivators include, e.g., applying putative modulator compounds tocells, in the presence or absence of a polypeptide or polynucleotide ofthe invention and then determining the functional effects on apolypeptide or polynucleotide of the invention activity. Samples orassays comprising a polypeptide or polynucleotide of the invention thatare treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of effect. Control samples (untreatedwith modulators) are assigned a relative activity value of 100%.Inhibition is achieved when the activity value of a polypeptide orpolynucleotide of the invention relative to the control is about 80%,optionally 50% or 25-1%. Activation is achieved when the activity valueof a polypeptide or polynucleotide of the invention relative to thecontrol is 110%, optionally 150%, optionally 200-500%, or 1000-3000%higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid, fattyacid, polynucleotide, oligonucleotide, etc. The test compound can be inthe form of a library of test compounds, such as a combinatorial orrandomized library that provides a sufficient range of diversity. Testcompounds are optionally linked to a fusion partner, e.g., targetingcompounds, rescue compounds, dimerization compounds, stabilizingcompounds, addressable compounds, and other functional moieties.Conventionally, new chemical entities with useful properties aregenerated by identifying a test compound (called a “lead compound”) withsome desirable property or activity, e.g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 Daltons and less than about 2500 Daltons, less than about2000 Daltons, between about 100 and about 1000 Daltons, or between about200 and about 500 Daltons.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

We have previously shown that NAP (NAPVSIPQ, SEQ ID NO:1) protectsneurons and glial cells through interaction with brain tubulin (Divinskiet al, J. Biol. Chem. 279, 28531-28538 (2004)) and stimulation oftubulin assembly to increase neurite outgrowth which is associated withmicrotubule assembly (Gozes and Spivak-Pohis, Curr Alzheimer Res, 3:197-199 (2006)). By affinity chromatography, NAP was also shown tospecifically interact with beta III tubulin (Divinski et al., J.Neurochem, 98, 973-984 (2006)). SAL has likewise been shown to conferneuroprotection (e.g., Gozes et al., 2000; Brenneman et al., 1998).Previously it had been thought that the eight amino acid NAP coresequence and the nine amino acid SAL core sequence could not be modifiedwithout loss of function. This application provides the firstdemonstration of peptides that have sequence similarities with the NAPand SAL core sequences, but that also have biological function, e.g.,promotion of survival of neuronal cells. NAP-like and SAL-like peptidemimetics were identified and are listed in Table 1 and 2 herein.Biological activity was found in at least two of the NAP-like peptidemimetics or SAL-like peptide mimetics: NATLSIHQ (SEQ ID NO:4) andSTPTAIPQ (SEQ ID NO:6). These compounds can be used as therapeuticmolecules for treatment of neurodegenerative diseases or disorders.

II. Design and Synthesis of Nap-Like and SAL-Like Peptide Mimetics

Modifications of polypeptides and peptides comprising the core NAP-likeor SAL-like peptide mimetic active site can be made, e.g., bysystematically adding one amino acid at a time to the N or C-terminus ofthe active core site and screening the resulting peptide for biologicalactivity, as described herein. In addition, the contributions made bythe side chains of various amino acid residues in such peptides can beprobed via a systematic scan with a specified amino acid, e.g., Ala.Polypeptides derived from the NAP-like or SAL-like peptide can also bemade.

Peptides with NAP-like and SAL-like sequences and properties can bederived from known proteins with sequences found in, e.g.,publicly-available databases. Examples include NCBI, OMIM,UniProtKB/Swiss-Prot, EMBOSS Pairwise Alignment Algorithms, ClustalW,Tcoffee, BLAST, RADAR, PROSITE, Phylogenetic Tree, and Selection.

NCBI (National Center for Biotechnology Information, USA) includesPubMed, a service of the U.S. National Library of Medicine that includesover 16 million citations from MEDLINE and other life science journalsfor biomedical articles back to the 1950s. PubMed includes links to fulltext articles and other related resources. NCBI also developed OMIM(Online Mendelian Inheritance in Man), a catalog of human genes andgenetic disorders. OMIM contains textual information, references, linksto MEDLINE and sequence records in the Entrez system, and links toadditional related resources at NCBI and elsewhere.

UniProtKB/Swiss-Prot is a manually annotated protein knowledgebasewhich, together with UniProtKB/TrEMBL, its computer-annotatedsupplement, gives access to all the publicly available proteinsequences. This database distinguishes itself from other proteinsequence databases by three distinct criteria: integration with otherdatabases, minimal redundancy and high annotation (such as; function ofthe protein, post-translational modification, domains and sites,secondary structure, quaternary structure, disease associated withdeficiencies in the protein sequence, variants, etc).

EMBOSS is “The European Molecular Biology Open Software Suite”. TheEMBOSS Pairwise Alignment tool is used to compare 2 sequences. ClustalWis a general purpose multiple sequence alignment program for DNA orproteins. It produces biologically meaningful multiple sequencealignments of divergent sequences, calculates the best match for theselected sequences, and lines them up so that the identities,similarities and differences can be seen. T-coffee is another optionsimilar to ClustalW.

Basic Local Alignment Search Tool (BLAST) finds regions of localsimilarity between sequences. The program compares nucleotide or proteinsequences to sequence databases and calculates the statisticalsignificance of matches. BLAST can be used to infer functional andevolutionary relationships between sequences as well as help identifymembers of gene families.

PROSITE is a database of protein families and domains that groupsproteins on the basis of similarities in their sequences into a limitednumber of families. Proteins or protein domains belonging to aparticular family generally share functional attributes and are derivedfrom a common ancestor. PROSITE currently contains patterns and profilesspecific for more than a thousand protein families or domains. Each ofthese signatures comes with documentation providing backgroundinformation on the structure and function of these proteins.

Phylogenetic tree relies on the NJ (Neighbour Joining) method of Saitouand Nei, which first calculates distances (percent divergence) betweenall pairs of sequence from a multiple alignment and then applies the NJmethod to the distance matrix. Selecton enables detecting of theselective forces at a single amino acid site. The ratio ofnon-synonymous (amino-acid altering) to synonymous (silent)substitutions, known as the Ka/Ks ratio, is used to estimate bothpositive and purifying selection at each amino acid site.

One of skill will recognize many ways of generating alterations in agiven nucleic acid sequence. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques (seeGiliman & Smith, Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734(1987)).

Most commonly, polypeptide sequences are altered by changing thecorresponding nucleic acid sequence and expressing the polypeptide.However, polypeptide sequences are also optionally generatedsynthetically using commercially available peptide synthesizers toproduce any desired polypeptide (see Merrifield, Am. Chem. Soc.85:2149-2154 (1963); Stewart & Young, Solid Phase Peptide Synthesis (2nded. 1984)).

One of skill can select a desired nucleic acid or polypeptide of theinvention based upon the sequences provided and upon knowledge in theart regarding proteins generally. Knowledge regarding the nature ofproteins and nucleic acids allows one of skill to select appropriatesequences with activity similar or equivalent to the nucleic acids andpolypeptides disclosed herein. The definitions section, supra, describesexemplar conservative amino acid substitutions.

Polypeptides are evaluated by screening techniques in suitable assaysfor the desired characteristic. For instance, changes in theimmunological character of a polypeptide can be detected by anappropriate immunological assay. Modifications of other properties suchas nucleic acid hybridization to a target nucleic acid, redox or thermalstability of a protein, hydrophobicity, susceptibility to proteolysis,or the tendency to aggregate are all assayed according to standardtechniques. Here, polypeptides that comprise a NAP-like or SAL-likemimetic active site are evaluated for biological activity, e.g.,reduction or inhibition of neuronal cell death.

More particularly, the small peptides of the present invention can bescreened by employing suitable assays and animal models known to thoseskilled in the art.

Using these assays and models, one of ordinary skill in the art canscreen a large number of NAP-like and SAL-like peptide mimetics inaccordance with the teachings of the present invention for those thatpossess the desired activity.

The peptides of the invention may be prepared via a wide variety ofwell-known techniques. Peptides of relatively short size are typicallysynthesized on a solid support or in solution in accordance withconventional techniques (see, e.g., Merrifield, Am. Chem. Soc.85:2149-2154 (1963)). Various automatic synthesizers and sequencers arecommercially available and can be used in accordance with knownprotocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis(2nd ed. 1984)). Solid phase synthesis in which the C-terminal aminoacid of the sequence is attached to an insoluble support followed bysequential addition of the remaining amino acids in the sequence is thepreferred method for the chemical synthesis of the peptides of thisinvention. Techniques for solid phase synthesis are described by Barany& Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A.; Merrifield et al 1963; Stewart et al. 1984). NAP andrelated peptides are synthesized using standard Fmoc protocols (Wellings& Atherton, Methods Enzymol. 289:44-67 (1997)).

In addition to the foregoing techniques, the peptides for use in theinvention may be prepared by recombinant DNA methodology. Generally,this involves creating a nucleic acid sequence that encodes the protein,placing the nucleic acid in an expression cassette under the control ofa particular promoter, and expressing the protein in a host cell.Recombinantly engineered cells known to those of skill in the artinclude, but are not limited to, bacteria, yeast, plant, filamentousfungi, insect (especially employing baculoviral vectors) and mammaliancells.

The recombinant nucleic acids are operably linked to appropriate controlsequences for expression in the selected host. For E. coli, examplecontrol sequences include the T7, trp, or lambda promoters, a ribosomebinding site and, preferably, a transcription termination signal. Foreukaryotic cells, the control sequences typically include a promoterand, preferably, an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, etc., and a polyadenylation sequence, and may includesplice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods. Such methods include, for example, thecalcium chloride transformation method for E. coli and the calciumphosphate treatment or electroporation methods for mammalian cells.Cells transformed by the plasmids can be selected by resistance toantibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo, and hyg genes.

Once expressed, the recombinant peptides can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, e.g., Scopes, PolypeptidePurification (1982); Deutscher, Methods in Enzymology Vol. 182: Guide toPolypeptide Purification (1990)). Optional additional steps includeisolating the expressed protein to a higher degree, and, if required,cleaving or otherwise modifying the peptide, including optionallyrenaturing the protein.

After chemical synthesis, biological expression or purification, thepeptide(s) may possess a conformation substantially different than thenative conformations of the constituent peptides. In this case, it ishelpful to denature and reduce the peptide and then to cause the peptideto re-fold into the preferred conformation. Methods of reducing anddenaturing peptides and inducing re-folding are well known to those ofskill in the art (see Debinski et al., J. Biol. Chem. 268:14065-14070(1993); Kreitman & Pastan, Bioconjug. Chem. 4:581-585 (1993); andBuchner et al., Anal. Biochem. 205:263-270 (1992)). Debinski et al., forexample, describe the denaturation and reduction of inclusion bodypeptides in guanidine-DTE. The peptide is then refolded in a redoxbuffer containing oxidized glutathione and L-arginine.

One of skill will recognize that modifications can be made to thepeptides without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion peptide. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

III. Functional Assays and Therapeutic Uses of Nap-Like and SAL-LikePeptide Mimetics

One method to determine biological activity of a NAP-like or SAL-likepeptide mimetic is to assay their ability to protect neuronal cells fromdeath. One such assay is performed using dissociated cerebral corticalcultures prepared as described (Brenneman & Gozes, J. Clin. Invest.97:2299-2307 (1996)). The test paradigm consists of the addition of atest peptide to cultures that are co-treated with tetrodotoxin (TTX).TTX produces an apoptotic death in these cultures and, thus, is used asa model substance to demonstrate efficacy against this “programmed celldeath” and all other means that produce this type of death mechanism.The duration of the test period is 5 days, and neurons are counted andidentified by characteristic morphology and by confirmation with animmunocytochemical marker for neurons: e.g., neuron specific enolase.Other cell based assays include assaying the ability of NAP-like orSAL-like peptides to promote survival of neuronal cells exposed to,e.g., beta-amyloid protein or high levels of ZnCl₂. These assays aredemonstrated in Example 2, herein. Neuronal cell survival promoted byNAP-like and SAL-like proteins can also be measured in the presence ofneurotoxins such as, gp120, the envelope protein from HIV andN-methyl-D-aspartic acid.

In another aspect, the present invention provides a method for reducingneuronal cell death, the method comprising contacting neuronal cellswith a NAP-like or SAL-like peptide mimetic in an amount sufficient toreduce neuronal cell death. In a further aspect, the NAP-like orSAL-like peptide mimetic comprises at least one D-amino acid within itsactive core site, preferably at the N-terminus and/or the C-terminus ofthe active core site. In another preferred aspect, each amino acid ofthe core NAP-like or SAL-like peptide is a D-amino acid. PreferredNAP-like or SAL-like peptide mimetics, include, e.g., NATLSIHQ (SEQ IDNO:4) and STPTAIPQ (SEQ ID NO:6).

NAP-like and SAL-like peptide mimetics of the present invention can beused in the treatment of neurological disorders and for the preventionof neuronal cell death. For example, NAP-like peptide mimetics of thepresent invention can be used to prevent the death of neuronal cellsincluding, but not limited to, spinal cord neurons, hippocampal neurons,cerebral cortical neurons and cholinergic neurons. More particularly,NAP-like and SAL-like peptide mimetics of the present invention can beused in the prevention of cell death associated with (1) gp120, theenvelope protein from HIV; (2) N-methyl-D-aspartic acid(excito-toxicity); (3) tetrodotoxin (blockage of electrical activity);and (4) β-amyloid peptide, a substance related to neuronal degenerationin Alzheimer's disease. Preferred NAP-like or SAL-like peptide mimetics,include, e.g., NATLSIHQ (SEQ ID NO:4) and STPTAIPQ (SEQ ID NO:6).

As such, the NAP-like and SAL-like peptide mimetics of the presentinvention can be used to reduce gp120-induced neuronal cell death byadministering an effective amount of an NAP-like peptide mimetic of thepresent invention to a patient infected with the HIV virus. The NAP-likeand SAL-like peptide mimetics of the present invention can also be usedto reduce neuronal cell death associated with excito-toxicity induced byN-methyl-D-aspartate stimulation, the method comprising contactingneuronal cells with an NAP-like and SAL-like peptide mimetic of thepresent invention in an amount sufficient to prevent neuronal celldeath. The NAP-like and SAL-like peptide mimetics of the presentinvention can also be used to reduce cell death induced by the β-amyloidpeptide in a patient afflicted or impaired with Alzheimer's disease, themethod comprising administering to the patient an NAP-like and SAL-likepeptide mimetic of the present invention in an amount sufficient toprevent neuronal cell death. The NAP-like and SAL-like peptide mimeticscan also be used to alleviate learning impairment produced bycholinergic blockage in a patient afflicted or impaired with Alzheimer'sdisease. For example, NAP-like and SAL-like peptide mimetics can be usedto improve short-term and/or reference memory in Alzheimer's patients.Preferred NAP-like or SAL-like peptide mimetics, include, e.g., NATLSIHQ(SEQ ID NO:4) and STPTAIPQ (SEQ ID NO:6).

Similarly, it is apparent to those of skill in the art that the NAP-likeand SAL-like peptide mimetics of the present invention can be used in asimilar manner to prevent neuronal cell death associated with a numberof other neurological diseases and deficiencies. Pathologies that wouldbenefit from therapeutic and diagnostic applications of this inventioninclude conditions (diseases and insults) leading to neuronal cell deathand/or sub-lethal neuronal pathology including, for example, thefollowing: diseases of central motor systems including degenerativeconditions affecting the basal ganglia (Huntington's disease, Wilson'sdisease, striatonigral degeneration, corticobasal ganglionicdegeneration), Tourette's syndrome, Parkinson's disease, progressivesupranuclear palsy, progressive bulbar palsy, familial spasticparaplegia, spinomuscular atrophy, ALS and variants thereof,dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplasticcerebellar degeneration, and dopamine toxicity; diseases affectingsensory neurons such as Friedreich's ataxia, diabetes, peripheralneuropathy, retinal neuronal degeneration; diseases of limbic andcortical systems such as cerebral amyloidosis, Pick's atrophy, Rettssyndrome; neurodegenerative pathologies involving multiple neuronalsystems and/or brainstem including Alzheimer's disease, AIDS-relateddementia, Leigh's disease, diffuse Lewy body disease, epilepsy, multiplesystem atrophy, Guillain-Barre syndrome, lysosomal storage disorderssuch as lipofuscinosis, late-degenerative stages of Down's syndrome,Alper's disease, vertigo as result of CNS degeneration; pathologiesassociated with developmental retardation and learning impairments, andDown's syndrome, and oxidative stress induced neuronal death;pathologies arising with aging and chronic alcohol or drug abuseincluding, for example, with alcoholism the degeneration of neurons inlocus coeruleus, cerebellum, cholinergic basal forebrain; with agingdegeneration of cerebellar neurons and cortical neurons leading tocognitive and motor impairments; and with chronic amphetamine abusedegeneration of basal ganglia neurons leading to motor impairments;pathological changes resulting from focal trauma such as stroke, focalischemia, vascular insufficiency, hypoxic-ischemic encephalopathy,hyperglycemia, hypoglycemia, closed head trauma, or direct trauma;pathologies arising as a negative side-effect of therapeutic drugs andtreatments (e.g., degeneration of cingulate and entorhinal cortexneurons in response to anticonvulsant doses of antagonists of the NMDAclass of glutamate receptor, peripheral neuropathies resulting from,e.g., chemotherapy treatments, and retinal damage from laser eyetreatments). NAP-like and SAL-like peptide mimetics of the presentinvention can also be used to treat autoimmune diseases, such asmultiple sclerosis and mental disorders, such as schizophrenia anddepression. Preferred NAP-like or SAL-like peptide mimetics, include,e.g., NATLSIHQ (SEQ ID NO:4) and STPTAIPQ (SEQ ID NO:6).

Thus, the NAP-like and SAL-like peptide mimetics that reduce neuronalcell death can be screened using the various methods described inInternational PCT Application Publication No. WO98/35042, filed Feb. 7,1997, and U.S. Pat. No. 6,613,740, filed Nov. 6, 1998. For example, itwill be readily apparent to those skilled in the art that using theteachings set forth above with respect to the design and synthesis ofNAP-like and SAL-like peptide mimetics and the assays described herein,one of ordinary skill in the art can identify other biologically activeNAP-like peptide mimetics comprising at least one D-amino acid withintheir active core sites. For example, Brenneman et al., Nature335:639-642 (1988), and Dibbern et al., J. Clin. Invest. 99:2837-2841(1997), teach assays that can be used to screen ADNF polypeptides thatare capable of reducing neuronal cell death associated with envelopeprotein (gp120) from HIV. Also, Brenneman et al., Dev. Brain Res.51:63-68 (1990), and Brenneman & Gozes, J. Clin. Invest. 97:2299-2307(1996), teach assays that can be used to screen NAP-like and SAL-likepeptide mimetics which are capable of reducing neuronal cell deathassociated with excito-toxicity induced by stimulation byN-methyl-D-aspartate. Other assays described in, e.g., International PCTApplication Publication No. WO98/35042 can also be used to identifyother biologically active NAP-like and SAL-like peptide mimetics.

Moreover, NAP-like and SAL-like peptide mimetics that reduce neuronalcell death can be screened in vivo. For example, the ability of NAP-likeand SAL-like peptide mimetics that can protect against learning andmemory deficiencies associated with cholinergic blockade can be tested.For example, cholinergic blockade can be obtained in rats byadministration of the cholinotoxin AF64A, and ADNF polypeptides can beadministered intranasally and the water maze experiments can beperformed (Gozes et al., Proc. Natl. Acad. Sci. USA 93:427-432 (1996)).Animals treated with efficacious NAP-like peptide mimetics would showimprovement in their learning and memory capacities compared to thecontrol.

Furthermore, the ability of NAP-like and SAL-like peptide mimetics thatcan protect or reduce neuronal cell death associated with Alzheimer'sdisease can be screened in vivo. For these experiments, apolipoprotein E(ApoE)-deficient homozygous mice can be used (Plump et al., Cell71:343-353 (1992); Gordon et al., Neuroscience Letters 199:1-4 (1995);Gozes et al., J. Neurobiol. 33:329-342 (1997)).

The ability of NAP-like and SAL-like peptide mimetics to inhibit immunecell proliferation, can be assayed as described in Offen et al. J Mol.Neurosci. 15(3):167-76 (2000) and International PCT ApplicationPublication No. WO04/060309, both of which describe the MOG-inducedchronic EAE mouse model and are herein incorporated by reference for allpurposes. The STOP protein-deficient mouse is an art accepted model ofschizophrenia can be used to assess anti-schizophrenia activity ofNAP-like and SAL-like peptide mimetics. See, e.g., Andrieux et al.,Genes & Develop., 16:2350-2364 (2002), which is herein incorporated byreference for all purposes. Anti-anxiety activity of NAP-like andSAL-like peptide mimetics can be assessed using a mouse model and theMorris water maze paradigm, disclosed at International PCT ApplicationPublication No. WO04/080957, which is herein incorporated by referencefor all purposes. Reduction of peripheral neurotoxicity by NAP-like andSAL-like peptide mimetics can be assessed using a rat model and rota-rodand plantar tests. See, e.g., International PCT Application PublicationNo. WO06/099739, which is herein incorporated by reference for allpurposes.

IV. Drug Discovery

The identification of tubulin as a NAP-interacting protein and thediscovery of NAP-like sequences in tubulin allows the use of tubulin andtubulin-derived peptides as targets for further drug discovery, e.g.,for the treatment of neuronal disorders such as neurodegenerativedisorders (e.g., Alzheimer's disease, AIDS-related dementia,Huntington's disease, and Parkinson's disease), cognitive deficits,peripheral neurotoxicity, motor dysfunctions, sensory dysfunctions,anxiety, depression, psychosis, conditions involving retinaldegeneration, disorders affecting learning and memory, orneuropsychiatric disorders, diseases related to neuronal cell death andoxidative stress, HIV-related dementia complex, stroke, head trauma,cerebral palsy, conditions associated with fetal alcohol syndrome, andautoimmune diseases, such as multiple sclerosis. Such therapeutics canalso be used in methods of enhancing learning and memory both pre- andpost-natally. Experiments can be carried out to find agents that bindthe same site as NAP using the intact tubulin structure and NAP as adisplacing agent (e.g., as described Katchalski-Katzir et al., BiophysChem. 100(1-3):293-305 (2003); Chang et al., J Comput Chem.24(16):1987-98 (2003)).

Preliminary screens can be conducted by screening for agents capable ofbinding to a polypeptide of the invention or tubulin, as at least someof the agents so identified are likely modulators binding activity. Thebinding assays usually involve contacting a polypeptide of the inventionwith one or more test agents and allowing sufficient time for theprotein and test agents to form a binding complex. Any binding complexesformed can be detected using any of a number of established analyticaltechniques. Protein binding assays include, but are not limited to,methods that measure co-precipitation, co-migration on non-denaturingSDS-polyacrylamide gels, and co-migration on Western blots (see, e.g.,Bennet and Yamamura, Neurotransmitter, Hormone or Drug Receptor BindingMethods, in Neurotransmitter Receptor Binding (Yamamura et al., eds.),pp. 61-89 (1985). The protein utilized in such assays can be naturallyexpressed, cloned or synthesized.

Agents that are initially identified by any of the foregoing screeningmethods can be further tested to validate the apparent activity.Preferably such studies are conducted with suitable animal models. Thebasic format of such methods involves administering a lead compoundidentified during an initial screen to an animal that serves as a modelfor humans and then determining if expression or activity of apolynucleotide or polypeptide of the invention is in fact upregulated.The animal models utilized in validation studies generally are mammalsof any kind. Specific examples of suitable animals include, but are notlimited to, primates, mice, and rats.

The agents tested as modulators of the polypeptides of the invention canbe any small chemical compound, or a biological entity, such as aprotein, sugar, nucleic acid, RNAi, or lipid. Typically, test compoundswill be small chemical molecules and peptides. Essentially any chemicalcompound can be used as a potential modulator or ligand in the assays ofthe invention, although most often compounds that can be dissolved inaqueous or organic (especially DMSO-based) solutions are used. Theassays are designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source toassays, which are typically run in parallel (e.g., in microtiter formatson microtiter plates in robotic assays). It will be appreciated thatthere are many suppliers of chemical compounds, including Sigma (St.Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.),Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.Modulators also include agents designed to reduce the level of mRNA ofthe invention (e.g. antisense molecules, ribozymes, DNAzymes and thelike) or the level of translation from an mRNA.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity, e.g.,tubulin binding. The compounds thus identified can serve as conventional“lead compounds” or can themselves be used as potential or actualtherapeutics. Libraries available for screening for small activemolecules include the Available Chemical Directory (ACD, 278,000compounds), ACD screening library (>1,000,000 compounds), CRC CombinedChemical Dictionary (˜350,000 compounds) Anisex (115,000 compounds)Maybridge (62,000 compounds) Derwent and NCI libraries.

V. Assays for Activity of Discovered Compounds

Additional drug discovery methods include screening for neuroprotectiveactivity. Such activity can be tested in classical tissue culture modelsof neuronal stress and survival as described, e.g., in Divinski et al.(2006) and Gozes et al. (2005). These assays are known in the art andfocus on the effect of test compounds on microtubule reorganization,neurite outgrowth, and protection from toxic factors.

In vivo assays to test neuroprotection in animal models are also knownin the art. Tests that measure effects of various test substances onmotor activity include the rotorod test, e.g., in rats. Olfactioncapacity can be used to measure the effect of test substances on sensoryactivity. Such assays are described, e.g., in U.S. App. Publication No.2006/0247168.

A well-established model for fetal alcohol syndrome can be used to testthe efficacy of test compounds (Webster et al., Neurobehav. Toxicol2:227-234 (1980)). This paradigm is a test for efficacy against severeoxidative stress produced from alcohol administration (Spong et al.,2001). This model allows for a rapid and relevant evaluation of agentsefficacious against severe oxidative stress as well as fetal alcoholsyndrome. To assess the protective effects of a test compound, thenumber of fetal demises can be determined

Experiments to test the protective effect of a test compound on retinalcells exposed to lasers, e.g., in conditions of laser surgery, aredescribed in U.S. Prov. App. No. 60,776,329. In brief, rats were exposedto laser photocoagulation and immediately treated either systemically orintravitreously with a protective compound. The animals were sacrificedand retinal tissue sections were observed for histological andmorphological abnormalities.

As discussed above, modulators of NAP-like and SAL-like peptide mimeticscan be assayed for ability to inhibit immune cell proliferation,anti-schizophrenia activity, anti-anxiety activity, and ability toreduce peripheral neurotoxicity

VI. Pharmaceutical Administration

The invention provides a number of neuroprotective NAP-like and SAL-likepeptide mimetics and compositions for pharmaceutical administration. Forexample, a pharmaceutical composition can comprise one of the NAP-likeor SAL-like peptide mimetics described herein, or more than one, incombination. Preferred NAP-like or SAL-like peptide mimetics, include,e.g., NATLSIHQ (SEQ ID NO:4) and STPTAIPQ (SEQ ID NO:6). Thepharmaceutical composition can include additional neuroprotectivecompounds, such as ADNF polypeptides, in combination with the NAP-likeor SAL-like peptide mimetic. Neuroprotective ADNF polypeptides includethose comprising NAP (SEQ ID NO:1) or SAL (SEQ ID NO:19). The NAP-likepeptide mimetic can comprise at least one D-amino acid, and as many asall of the amino acids can be D-chirality. In some embodiments, theadditional neuroprotective peptide has at least one, and as many as all,D-amino acids.

The pharmaceutical compositions of the present invention are suitablefor use in a variety of drug delivery systems. Peptides that have theability to cross the blood brain barrier can be administered, e.g.,systemically, nasally, etc., using methods known to those of skill inthe art. Larger peptides that do not have the ability to cross the bloodbrain barrier can be administered to the mammalian brain viaintracerebroventricular (ICV) injection or via a cannula usingtechniques well known to those of skill in the art (see, e.g., Motta &Martini, Proc. Soc. Exp. Biol. Med. 168:62-64 (1981); Peterson et al.,Biochem. Pharamacol. 31:2807-2810 (1982); Rzepczynski et al., Metab.Brain Dis. 3:211-216 (1988); Leibowitz et al., Brain Res. Bull.21:905-912 (1988); Sramka et al., Stereotact. Funct. Neurosurg. 58:79-83(1992); Peng et al., Brain Res. 632:57-67 (1993); Chem et al., Exp.Neurol. 125:72-81 (1994); Nikkhah et al., Neuroscience 63:57-72 (1994);Anderson et al., J. Comp. Neurol. 357:296-317 (1995); and Brecknell &Fawcett, Exp. Neurol. 138:338-344 (1996)).

Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences (17th ed. 1985)). In addition, for abrief review of methods for drug delivery, see Langer, Science249:1527-1533 (1990). Suitable dose ranges are described in the examplesprovided herein, as well as in International PCT Application PublicationNo. WO 9611948.

As such, the present invention provides for therapeutic compositions ormedicaments comprising one or more of the polypeptides describedhereinabove in combination with a pharmaceutically acceptable excipient,wherein the amount of polypeptide is sufficient to provide a therapeuticeffect.

In a therapeutic application, the polypeptides of the present inventionare embodied in pharmaceutical compositions intended for administrationby any effective means, including parenteral, topical, oral, nasal,pulmonary (e.g. by inhalation), systemic, or local administration. Forparenteral administration, the pharmaceutical compositions areadministered e.g., intravenously, subcutaneously, intradermally, orintramuscularly. Nasal pumps, topical patches, and eye drops can also beused.

Thus, the invention provides compositions for parenteral administrationthat comprise a solution of polypeptide, as described above, dissolvedor suspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used including, for example, water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques or, they may be sterile filtered. The resultingaqueous solutions may be packaged for use as is or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionsincluding pH adjusting and buffering agents, tonicity adjusting agents,wetting agents and the like, such as, for example, sodium acetate,sodium lactate, sodium chloride potassium chloride, calcium chloride,sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be usedthat include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the polypeptides are preferably supplied infinely divided form along with a surfactant and propellant. Thesurfactant must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. A carrier can also be included, as desired, as with, e.g.,lecithin for intranasal delivery. An example includes a solution inwhich each milliliter included 7.5 mg NaCl, 1. 7 mg citric acidmonohydrate, 3 mg disodium phosphate dihydrate and 0.2 mg benzalkoniumchloride solution (50%) (Gozes et al., J Mol. Neurosci. 19(1-2):167-70(2002)).

In therapeutic applications, the polypeptides of the invention areadministered to a patient in an amount sufficient to reduce or eliminatesymptoms of neurodegenerative disorders, cognitive deficits, and otherconditions, or to enhance learning and memory. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” Amountseffective for this use will depend on, for example, the particularpolypeptide employed, the type of disease or disorder to be prevented,the manner of administration, the weight and general state of health ofthe patient, and the judgment of the prescribing physician.

For example, an amount of polypeptide falling within the range of a 100ng to 10 mg dose given intranasally once a day (e.g., in the evening)would be a therapeutically effective amount. Alternatively, dosages maybe outside of this range, or on a different schedule. For example,dosages may range from 0.0001 mg/kg to 10,000 mg/kg, and will preferablybe about 0.001 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 50 mg/kg or 500 mg/kgper dose. Doses may be administered hourly, every 4, 6 or 12 hours, withmeals, daily, every 2, 3, 4, 5, 6, or 7 days, weekly, every 2, 3, 4weeks, monthly or every 2, 3 or 4 months, or any combination thereof.The duration of dosing may be single (acute) dosing, or over the courseof days, weeks, months, or years, depending on the condition to betreated. Those skilled in the art can determine the suitable dosage, andmay rely on preliminary data reported in Gozes et al., 2000; Gozes etal., 2002; Bassan et al. 1999; Zemlyak et al., Regul. Pept. 96:39-43(2000); Brenneman et al., Biochem. Soc. Trans. 28: 452-455 (2000);Erratum Biochem Soc. Trans. 28:983; Wilkemeyer et al. Proc. Natl. Acad.Sci. USA 100:8543-8548 (2003); Alcalay et al., Neurosci Lett. 361:128-31(2004); and Gozes et al., CNS Drug Rev., 11(4):353-68 (2005).

EXAMPLES Example 1 Search for NAP-Like and SAL-Like Sequences

A bio-informatics search was launched to address whether there areNAP-like or SAL-like sequences in other proteins that provideneuroprotection (e.g., through interaction with microtubules) andwhether there are tubulin specific sequences that resemble NAP andprovide neuroprotection.

The NAP and SAL sequences were submitted to a number of different searchengines: NCBI, OMIM, UniProtKB/Swiss-Prot, EMBOSS Pairwise AlignmentAlgorithms, ClustalW, T-coffee, BLAST, RADAR, PPSearch, PROSITE,Phylogenetic Tree, and Selecton.

In the search for human tubulin proteins, the field descriptions tubulinand the boolean operators for Homo sapiens organism were used inUniProtKB/Swiss-Prot. Blosum62 with water alignment was used in EMBOSSin order to find the best region of similarity between two sequences.Multiple alignments were obtained from ClustalW, with further use of theJalview editor.

For BLAST, and the similar programs RADAR and PPSearch, human beta3tubulin and its orthologs were used as a query. For Selecton, the CDS oftubulin and the 12 ortholog organisms were submitted in FASTA format asan input file.

The results are summarized below and in Table 1. Structural elementswithin tubulin that are important for protein-protein interaction andGTP binding show significant homology to NAP:

(SEQ ID NO: 2) NAVLSIHQ-Tubulin beta1 (SEQ ID NO: 3)NATLSVHQ-Tubulin beta2 (SEQ ID NO: 4) NATLSIHQ-Tubulin beta3

NCBI protein Access Numbers for the various tubulin subunits:

Tubulin beta1 Q9H4B7 Tubulin beta 2a Q13885 Tubulin beta 2b Q9BVA1Tubulin beta 2c P68371 Tubulin beta 3 Q13509 Tubulin beta 4 P04350Tubulin beta 5 P07437 Tubulin beta 6 Q9BUF5

The sequence s NAVLSIHQ (SEQ ID NO:2), NATLSVHQ (SEQ ID NO:3), andNATLSIHQ (SEQ ID NO:4), found in tubulin beta1, beta2, and beta3,respectively, but not alpha tubulin. The sequence runs from amino acids184-191. This sequence overlaps with an area that is hypothesized to beimportant in the longitudinal contacts between beta and alpha tubulinwithin a microtubule, i.e., it sits at a relatively exposed area at thetop of the molecule which becomes hidden upon dimerization. The sequenceis also close to the GTP binding pocket of beta-tubulin, particularlythe area associated with ribose binding (Nogales and Wang (2006) CurrOpin Cell Biol, 18, 179-184; Nogales and Wang (2006) Curr Opin StructBiol, 16, 221-229.

The homology is >50% but there is no preservation of the two prolinesfound in NAP. Given that prolines are often associated withprotein-protein interactions, it is likely that NAPVSIPQ (SEQ ID NO:1)has additional protein binding or protein interaction/disruptionactivities while still having some intrinsic association withmicrotubules.

Other sequences with increased homology to NAPVSIPQ (SEQ ID NO:1)include STPTAIPQ (SEQ ID NO:6) (accession number Q7KZS6), which includesboth a tubulin segment and a segment relating to a G-protein coupledreceptor from the rhodopsin family. The latter has similarity tomelanocortin 1 receptor associated with pigmentation.

Additional sequence similarities were observed with key proteins suchas: citrate lyase (Table 1). ATP citrate-lyase is the primary enzymeresponsible for the synthesis of cytosolic acetyl-CoA in many tissues.It has a central role in de novo lipid synthesis. In nervous tissue itmay be involved in the biosynthesis of acetylcholine (by similarity).

TABLE 1 NAP (NAPVSIPQ) sequence homologies Query 1 NAPVSIPQ 8(SEQ ID NO: 1) DNA primase Acidovorax sp. JS42 NXPVSIPQ (SEQ ID NO: 14)Sbjct 3 NTPVSIPQ 10 (SEQ ID NO: 7) Query 2 APVSIPQ 8 (SEQ ID NO: 8)citrate lyase, alpha subunit Thermosinus APVSIPQ (SEQ ID NO: 8)carboxydivorans Nor1 Sbjct 217 APVSIPQ 223 (SEQ ID NO: 8) Query 1NAPVSIPQ 8 (SEQ ID NO: 1) putative citrate lyase alpha subunit NXP+SIPQ(SEQ ID NO: 15) Streptococcus pyogenes Sbjct 215 NTPISIPQ 222(SEQ ID NO: 9) str. Manfredo Query 1 NAPVSIPQ 8 (SEQ ID NO: 1)citrate lyase alpha subunit Lactobacillus NXP+SIPQ (SEQ ID NO: 15)paracasei Sbjct 158 NTPISIPQ 165 (SEQ ID NO: 9) Query 1 NAPVSIPQ 8(SEQ ID NO: 1) Chain A, Crystal Structure Of The Putative NXP+SIPQ(SEQ ID NO: 15) Alfa Subunit Of Citrate Sbjct 215 NTPISIPQ 222(SEQ ID NO: 9) Chain B, Crystal Structure Of The PutativeAlfa Subunit Of Citrate Lyase In Complex With Citrate FromStreptococcus Mutans, Northeast Structural Genomics Target Smr12(Casp Target) Query 1 NAPVSIPQ 8 (SEQ ID NO: 1)Citrate lyase alpha chain/Citrate CoA- NXP+SIPQ (SEQ ID NO: 15)transferase Streptococcus Sbjct 215 NTPISIPQ 222 (SEQ ID NO: 9)pyogenes MGAS10270] Query 1 NAPVSIPQ 8 (SEQ ID NO: 1)citrate lyase alpha subunit Enterococcus NXP+SIPQ (SEQ ID NO: 15)faecalis Sbjct 215 NTPISIPQ 222 (SEQ ID NO: 9) Query 1 NAPVSIP 7(SEQ ID NO: 10) RING finger domain protein Neosartorya NAPVSIP(SEQ ID NO: 10) fischeri NRRL 181 Sbjct 102 NAPVSIP 108 (SEQ ID NO: 10)Query 2 APVSIPQ 8 (SEQ ID NO: 8) linear gramicidin synthetase subunit DAPVSIPQ (SEQ ID NO: 8) Mycobacterium avium 104 Sbjct 453 APVSIPQ 459(SEQ ID NO: 8) Query 2 APVSIPQ 8 (SEQ ID NO: 8) PstA Mycobacterium aviumAPVSIPQ (SEQ ID NO: 8) Sbjct 453 APVSIPQ 459 (SEQ ID NO: 8) Query 2APVSIPQ 8 (SEQ ID NO: 8) PstA Mycobacterium avium subsp. APVSIPQ(SEQ ID NO: 8) paratuberculosis K-10 Sbjct 461 APVSIPQ 467(SEQ ID NO: 8) Query 1 NAPVSIPQ 8 (SEQ ID NO: 1)glucose repression mediator protein Pichia NAPV++PQ (SEQ ID NO: 16)stipitis CBS 6054 Sbjct 760 NAPVAVPQ 767 (SEQ ID NO: 11) Query 1NAPVSIPQ 8 (SEQ ID NO: 1) adhesin family protein Granulibacter NAXVSIPQ(SEQ ID NO: 17) bethesdensis CGDNIH1 Sbjct 73 NARVSIPQ 80(SEQ ID NO: 12) Query 1 NAPVSIPQ 8 (SEQ ID NO: 1)cation efflux family protein Pseudomonas +APVS+PQ (SEQ ID NO: 18)fluorescens Pf-5 Sbjct 314 DAPVSVPQ 321 (SEQ ID NO: 13)

TABLE 2 SAL (SALLRSIPA) sequence homologies Query 2 ALLRSIPA 9(SEQ ID NO: 20) phosphatidylinositol glycan, class G, Danio ALLRSIPA(SEQ ID NO: 20) rerio Sbjct 614 ALLRSIPA 621 (SEQ ID NO: 20) Query 2ALLRSIPA 9 (SEQ ID NO: 20) heat shock protein 60 Salmo salar ALLRSIPA(SEQ ID NO: 20) Sbjct 53 ALLRSIPA 60 (SEQ ID NO: 20) Query 2 ALLRSIP 8(SEQ ID NO: 21) oligopeptide/dipeptide ABC transporter, ALLRSIP(SEQ ID NO: 21) ATPase subunit Thermotoga petrophila Sbjct 259 ALLRSIP265 (SEQ ID NO: 21) RKU-1 Query 2 ALLRSIPA 9 (SEQ ID NO: 20)oligopeptide/dipeptide ABC transporter, A+LRSIPA (SEQ ID NO: 28)ATPase subunit Burkholderia phymatum Sbjct 346 AMLRSIPA 353(SEQ ID NO: 22) STM815 Query 2 ALLRSIPA 9 (SEQ ID NO: 20)oligopeptide/dipeptide ABC transporter, ALLR+IPA (SEQ ID NO: 29)ATPase subunit Burkholderia phymatum Sbjct 254 ALLRAIPA 261(SEQ ID NO: 23) STM815 Query 2 ALLRSIP 8 (SEQ ID NO: 21)ABC peptide transporter, ATP-binding ALLRSIP (SEQ ID NO: 21)component Rhodococcus sp. RHA1 Sbjct 272 ALLRSIP 278 (SEQ ID NO: 21)Query 1 SALLRSIP 8 (SEQ ID NO: 24) similar to ATPase, H+transporting, V1 SALLR+IP (SEQ ID NO: 30)subunit E-like 2 isoform 2 Rattus norvegicus Sbjct 124 SALLRAIP 131(SEQ ID NO: 25) Query 2 ALLRSIPA 9 (SEQ ID NO: 20)glucose inhibited division protein A A+LRSIPA (SEQ ID NO: 28)Roseiflexus castenholzii Sbjct 366 AMLRSIPA 373 (SEQ ID NO: 22)DSM 13941 Query 2 ALLRSIPA 9 (SEQ ID NO: 20)glucose inhibited division protein A A+LRSIPA (SEQ ID NO: 28)Chloroflexus aggregans DSM 9485 Sbjct 346 AMLRSIPA 353 (SEQ ID NO: 22)Query 2 ALLRSIPA 9 (SEQ ID NO: 20) glucose inhibited division protein AA+LRSIPA (SEQ ID NO: 28) Herpetosiphon aurantiacus Sbjct 346 AMLRSIPA353 (SEQ ID NO: 22) ATCC 23779 Query 2 ALLRSIPA 9 (SEQ ID NO: 20)Glucose-inhibited division protein A A+LRSIPA (SEQ ID NO: 28)Roseiflexus sp. RS-1 Sbjct 364 AMLRSIPA 371 (SEQ ID NO: 22) Length = 679Query 1 SALLRSIP 8 (SEQ ID NO: 24) PAS/PAC sensor signal transductionSALLR+IP (SEQ ID NO: 30) histidine kinase Stigmatella aurantiaca Sbjct288 SALLRAIP 295 (SEQ ID NO: 25) DW4/3-1 Query 2 ALLRSIP 8(SEQ ID NO: 21) regulatory protein, LuxR Mariprofundus ALLRSIP(SEQ ID NO: 21) ferrooxydans PV-1 Sbjct 312 ALLRSIP 318 (SEQ ID NO: 21)Query 2 ALLRSIPA 9 (SEQ ID NO: 20) Tetratricopeptide TPR_2 HerpetosiphonALLR+IPA (SEQ ID NO: 29) aurantiacus ATCC 23779 Sbjct 189 ALLRTIPA 196(SEQ ID NO: 26) Query 2 ALLRSIPA 9 (SEQ ID NO: 20)coenzyme F390 synthetase/phenylacetyl- ALLRS+PA (SEQ ID NO: 31)CoA ligase Methanoculleus marisnigri JR1 Sbjct 406 ALLRSVPA 413(SEQ ID NO: 27) Query 2 ALLRSIP 8 (SEQ ID NO: 21)metal dependent phosphohydrolase ALLRSIP (SEQ ID NO: 21)Acidobacteria bacterium Ellin345 Sbjct 134 ALLRSIP 140 (SEQ ID NO: 21)

Example 2 Assays for Neuroprotective Activity

NATLSIHQ (SEQ ID NO:4) and STPTAIPQ (SEQ ID NO:6) are NAP-like peptides.The effect of these peptides on astrocyte and neuronal survivalfollowing ZnCl₂ and beta-amyloid intoxication were tested.

A. Methods:

1. Cerebral Cortical Astrocytes

Cell cultures were prepared as previously described (McCarthy K D, deVellis J., J. Cell Biol., 85:890-902 (1980); Gozes I et al., J.Pharmacol. Exp. Ther., 257:959-66 (1991)). Newborn mice (Harlan BiotechIsrael Ltd., Rehovot, Israel) were sacrificed by decapitation and thebrain was removed. The cortex was dissected and meninges were removed.The tissue was minced with scissors and placed in Hank's balanced saltssolution X1 (HBSS, Biological Industries, Beit Haemek, Israel), 15 mMHEPES Buffer pH 7.3 (Biological Industries, Beit Haemek, Israel) and0.25% trypsin (Biological Industries, Beit Haemek, Israel) in anincubator at 37° C. 10% CO₂ for 20 minutes. The cells were then placedin 8 ml of solution D1 containing 10% heat inactivated fetal calf serum(Biological Industries, Beit Haemek, Israel), 0.1% gentamycin sulphatesolution (Biological Industries, Beit Haemek, Israel) and 0.1%penicillin-streptomycin-nystatin solution (Biological Industries, BeitHaemek, Israel) in Dulbecco's modified Eagle's medium (DMEM, Sigma,Rehovot, Israel). The cells were allowed to settle, and were thentransferred to a new tube containing 2.5 ml of D1 and triturated using aPasteur pipette. The process was repeated twice more. Once all the cellswere suspended, cell density was determined using a hemocytometer(Neubauer improved, Germany) and 1×10⁶ cells/15 ml D1 were inoculatedinto each 75 cm² flask (Corning, Corning, N.Y., USA). Cells wereincubated at 37° C. 10% CO₂. The medium was changed after 24 hours andcells were grown until confluent (one week).

2. Cerebral Cortical Astrocyte Cell Subcultures

The flasks containing the cerebral cortical astrocytes were shaken todislodge residual neurons and oligodendrocytes that may be present.Flasks were then washed with 10 ml cold HBSSx1, HEPES15 mM. 5 mlversene-trypsin solution (BioLab, Jerusalem, Israel) was added to eachflask and the flasks were incubated at room temperature for 5 minutes toremove astrocytes. The flasks were then shaken to dislodge the cells.The versene-trypsin solution was neutralized with 5 ml D1. The cellsuspension was collected and centrifuged at 100 g for 10 minutes. Thesupernatant was removed and the cells resuspended in D1. The cells wereplated in 96 well plates (Corning, Corning, N.Y., USA) (each flask to 2plates) and incubated until confluent at 37° C. 10% CO₂.

3. Mixed Neuroglial Cultures

Newborn rats were used to prepare cerebral cortical astrocytes cellcultures as described above. After suspending the cells in D1, they werecentrifuged at 100 g for 5 minutes and the supernatant discarded. Thecell pellet was resuspended in solution D2 containing 5% heatinactivated horse serum (Biological Industries, Beit Haemek, Israel),0.1% gentamycin, 0.1% penicillin-streptomycin-nystatin, 1% N3 (definedmedium components essential for neuronal development in culture, (RomijnH J, Brain Res., 254:583-9 (1981)]), 15 μg/ml 5′-fluoro-2-deoxyuridine(FUDR, Sigma, Rehovot, Israel), and 3 μg/ml uridine (Sigma, Rehovot,Israel) in DMEM. Cells were counted in a hemocytometer, diluted in D2and 17,000 cells/well/96 well plate were seeded on 8-day-old astrocytesprepared as described above. The medium was changed the next day to D2without FUDR and uridine. Cells were allowed to grow for one week at 37°C. 10% CO₂ before experiments were performed.

4. MAP2 Assay

Neuronal survival in neuroglial cultures following beta-amyloidintoxication was assayed using the neuron specific antibody, MAP2. Oneweek after the preparation of the mixed neuroglial cultures, the cellgrowth medium was aspirated and fresh D2 medium was added to the cells.0.25 μM beta-amyloid 1-42 (American Peptide Company, Sunnyvale, Calif.,USA), dissolved in water and allowed to aggregate for at least two weeksin 37° C., was added to each well together with ascending concentrationsof either NATLSIHQ (SEQ ID NO:4) or STPTAIPQ (SEQ ID NO:6) from 10⁻¹⁹ Mto 10⁻⁵ M. The cells were incubated for 5 days in 10% CO₂ at 37° C.

5 days after the addition of beta-amyloid and the peptide, the cellswere fixed by removing the media from each well and the addition of coldmethanol. The cells were left in the refrigerator overnight. The cellswere immunostained with anti-MAP2 as previously described (Brooke S M etal., Neurosci. Lett., 267:21-4 (1999)): the methanol was removed and thecells were washed 4 times with phosphate buffered saline (PBS). Blockingfor non-specific antibody binding was performed by incubating the cellsin 5% non-fat milk in PBS overnight at 4° C. The blocking solution wasthen removed and anti-MAP2 (1:1000; Sigma, Rehovot, Israel) was added toeach well. The cells were incubated for 30 minutes at room temperature,followed by 4 washes with PBS. Biotinylated anti-mouse IgG (1:200,Vector Laboratories, Burlingame, Calif., USA) was then added to eachwell, and the cells were incubated for 30 minutes at room temperaturefollowed by 4 washes with PBS. The cells were incubated at roomtemperature for 30 minutes with the ABC reagent (Vector Laboratories,Burlingame, Calif., USA) prepared according to the manufacturer'sprotocol and then washed 4 times with PBS. ABTS reagent, preparedaccording to the manufacturer's protocol (Vector Laboratories,Burlingame, Calif., USA) was then added to each well and the cells wereincubated for 20 minutes in the dark at room temperature. The plateswere read in an ELISA plate reader at 405 nm. As blanks, wellscontaining untreated cells and no primary antibody were used.

5. MTS Assay

The survival of astrocytes following intoxication with ZnCl₂ was testedusing the MTS assay. One week after sub-culturing the astrocytes into96-well plates, the astrocyte growth medium was aspirated and freshmedium containing 200 μM ZnCl₂ and ascending concentrations of NATLSIHQ(SEQ ID NO:4) or STPTAIPQ (SEQ ID NO:6) (concentration range: 10⁻¹⁶-10⁻⁷M) was added to the cells. The cells were incubated for 4 hours in 10%CO₂ at 37° C., followed by an MTS assay using Celltiter 96 Aqueousnon-radioactive cell proliferation assay (Promega, Madison, Wis., USA)which was performed according to the manufacturer's instructions andread in an ELISA plate reader at 490 nm.

B. Results:

Results are shown in FIGS. 1 and 2 and in Table 3, below. Both peptideswere active in the neuroprotection assays. The efficacy of NATLSIHQ (SEQID NO:4) was greater than that of STPTAIPQ (SEQ ID NO:6) in assays forsurvival of both neuroglial cells and astrocytes.

TABLE 3a summary of the effective concentrations of the tested peptides on astrocyteand neuronal survival. Peptide: Neurons (25 μM beta-amyloid)Astrocytes (200 μM ZnC1₂) STPTAIPQ (SEQ ID NO: 6) 10⁻¹³, 10⁻⁵ (p < 0.05)10⁻⁷ (p < 0.05) NATLSIHQ 10⁻¹⁷, 10⁻¹³, (p < 0.005) 10⁻¹⁰ (p < 0.05)(SEQ ID NO: 4) 10⁻¹⁹ 10⁻¹⁵ 10⁻⁹ (p < 0.05) 10⁻¹², 10⁻⁸ (p < 0.005)10⁻⁷ (p < 0.0005)

Example 3 The Effect of NAPVIPQ and NATLSIHQ on Tau PathologicalAggregation Leading to Neurofibrillary Tangle Formation

VQIVYK aggregation: Tau is a highly soluble protein. The unfoldedprotein lacks a defined 3D structure. Its main role is stabilization ofmicrotubules in neuronal axons. Tau contains three or four microtubulebinding repeats. ³⁰⁶VQIVYK³¹¹ is a peptide derived from the beginning ofthe third microtubule binding repeat of tau, which is present in all tauvariants. This sequence was found to be important for the aggregation oftau into paired helical filaments (PHFs), which aggregate to make thetangles found in Alzheimer's disease and related disorders.

It is hypothesized that inhibition of tau aggregation will constitutefuture therapeutics. The aim of this study was to compare NAPalpha-aminoisobutyric acid (where the prolines in NAPVSIPQ weresubstituted with alpha-aminoisobutyric acid) with NAPVSIPQ containingprolines in an in vitro tau-like aggregation assay.

In vitro aggregation assay was performed in the presence of polyglutamicacid (or heparin), VQIVYK aggregates were further detected by ThioflavinS (excitation 485 nm and emission 535) with emission intensity greatlyincreasing.

1. Calibration of VQIVYK Aggregation Conditions

First, different concentrations of polyglutamic acid (0,100 μM, 250 μM,400 μM) VQIVYK and either sodium acetate (NH₄Ac) 50 mM pH 6.5 or MOPS 20mM pH 6.5 and Thioflavin S 5 μM were mixed together and incubated atroom temperature. The extent of aggregation was read at excitation 485nm and emission 535 nm using the infinite 200 system with the Magellanprogram. Optimal aggregation conditions were found to be at 7 days with100 μM VQIVYK, 250 μM polyglutamate and 20 mM MOPS pH 6.5 (see FIG. 3).

2. The Effect of the Peptides NAPVSIPQ (NAP) and NATLSIHQ NAP (isoNAP)on the Extent of VQIVYK Aggregation

As shown in FIG. 4, the peptides were added at a range of concentrations(10⁻¹⁷ M-10⁻⁹ M) and the extent of aggregation of 100 μM VQIVYK wastested in the presence of polyglutamate 250 μM in MOPS 20 μM, pH 6.5 for7 days.

In order to avoid reading self peptide aggregation as VQIVYKaggregation, for each peptide concentration, the fluorescence of thepeptide solution without VQIVYK was subtracted from the fluorescence ofeach peptide concentration containing VQIVYK. NATLSIHQ seems to besuperior to NAP in terms of inhibition of tau aggregation addingadditional claims and covering protein aggregation diseases.

REFERENCES

-   1. Friedhoff et al., Biochemistry, 1998, 37, 10223-10230.-   2. Perez et al., Journal of Neurochemistry, 2007, 103, 1447-1460.-   3. von Bergen et al., PNAS, 2000, 97, 5129-5134.

Example 4 The Effect of Treatment with NAT on Learning and Memory in TauTransgenic Mice: an Animal Model for Human Tauopathy Materials andMethods Animals

The mouse model, used in the current study, was previously described(Ramsden et al., (2005) J Neurosci, 25, 10637-10647). TherTg(tauP301L)-4510 mouse (designated as Tau-Tg below) expresses thehuman 4-repeat Tau with the P301L mutation (4R0N) associated withfrontotemporal dementia and Parkinsonism linked to chromosome 17.

In this mouse model, levels of several soluble phosphorylated tauspecies were highest at 1 month relative to later time points, thismaterial was cleared by 3 months, while heat shock protein expressionincreased with normal aging. This process was accelerated in rTg4510mice. Moreover, endogenous mouse tau turnover was slowed in response tohuman tau over-expression, and this endogenous tau adopteddisease-related properties (Dickey et al., (2009) Am J Pathol, 174,228-238). The onset of memory deficit was first observed at 2.5 monthsand was significant at 4 months. Mature neurofibrillary tangles,detected by Bielschowsky silver stain, appeared at 4 months andsignificant neuronal loss was estimated by stereology at 5.5 months(Ramsden et al., (2005) J Neurosci, 25, 10637-10647).

The experiment included three groups: Tau-Tg female mice, 10-month-old,treated by intranasal administration of NAT 2 μg/5 μl/mouse/day (n=5) orvehicle (SW/mouse/day) (n=6), and as control, non-Tg female littermatestreated by vehicle (SW/mouse/day) (n=7).

NAT (NATLSIHQ) Administration

NAT was dissolved in a vehicle solution, in which each milliliterinclude 7.5 mg of NaCl, 1.7 mg of citric acid monohydrate, 3 mg ofdisodium phosphate dehydrate, and 0.2 mg of benzalkonium chloridesolution (50%). 5 μl of NAT or vehicle solution (DD) were administeredintranasally.

Treatment started at 9 months of age and continued daily for a period of5 weeks. At each test NAT was applied 1 h before the test begun.

Comparative analysis between vehicle-treated transgenic andvehicle-treated non-Tg mice allowed evaluation of the pathologyassociated with the expression of the human mutant tau. By comparingTau-Tg NAT-treated mice and vehicle-treated mice peptide efficacy wastested.

All mice were weighed at the beginning and end of the experiment andwhole brain weight was measured before the brain dissection.

Behavioral Testing—Morris Water Maze (MWM)

Each mouse was placed in a pool of water that is colored opaque withpowdered non-fat milk, where it must swim to a hidden escape platform.The position of the platform was altered between days but remainedconstant within each day.

Test conditions: Pool diameter—on days 1-3: 80 cm and on days 4-5: 140cm. Platform-clear plaxiglass, 12 cm in diameter, 2 cm below the surfaceof the water. Water temperature—22° C.-23° C., Room temperature—26-28°C.

Experimental procedure: Mice were treated with NAT or vehicle and thenhabituated for 1 hour in the experiment room. The tested mouse wasplaced on the platform for 30 seconds followed by 2 sequential trialswith a cut-off of 90 seconds and an Intra Experimental Interval (IEI) of30 seconds in which it stayed on the platform. The time required forreaching the platform in each trial and the path lengths were measured.

On the fifth day, two additional tests were taken after the second dailytrial:

1. Probe test—The platform was removed from the maze. The mouse wasreleased at the same place in the pool as on the prior trial and thetime the mouse spent in the quarter in which the platform was situatedon the prior trial was recorded.

2. Visible platform test—In order to verify that all mice are capable ofseeing the platform was placed in the center of the pool, 1 cm above thewater surface. The mice passed the visible platform test.

Biochemical Analysis

Mouse brain tissue was rapidly dissected and quickly separated into fourdifferent brain sections: cortex, hippocampus, cerebellum and rest ofthe brain. Brains were kept frozen at −80° C. for further biochemicalanalysis. Total levels of nuclear ADNP were analyzed by immunoblotting.Cerebral cortex samples (−50 mg each) were homogenized and cytoplasmicand nucleus proteins were separated using lysis buffer (20 mM TRIS HClpH 7.7, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.2% NP-40) and extractionbuffer (10 mM TRIS HCl pH 7.7, 0.1 mM EDTA, 1.5 mM MgCl₂, 20% Glycerol,1.61gr NaCl). Protein amount was estimated and corrected by using theBradford assay and then separated by electrophoresis on 12%polyacrylamide gels containing SDS (Shiryaev et al., (2009) NeurobiolDis, 34, 381-388). Western blot analyses were performed by applyingbrain protein samples onto two gels. Each gel had sample representationfrom each one of the three groups. The proteins were transferred tonitrocellulose filter and immunostained with ADNP specific antibody (BDBioscience, 1/300). Proteins were visualized using enhancedchemiluminescence reagents, followed by exposure onto hyperfilm (Kodak)(Mandel and Gozes (2007) J Biol Chem, 282, 34448-34456). Protein bandson hyperfilm were quantified using photochromatography analysis. TheADNP amount in each band was calculated as its percentage from the totalamount of all bands. ADNP amounts of each group were averaged.

Statistical Analyses

Results are described as means±standard error (S.E.). Initialstatistical analyses compared only two groups among the three andincluded two-tailed indipendent t-tests. P values of 0.05 were deemedstatistically significant. Additional statistical analyses wereperformed using One-way ANOVA to compare the three experimental groupsfollowed by Tukey's Honestly Significantly Different (HSD) post-hoctest.

Results Tau Transgenic Mice Exhibited Deficit in Spatial Learning andWorking Memory

At the third experimental week, mice treated daily with NAT or vehiclewere subjected to two daily tests for five executive days in the Morriswater maze (MWM) that evaluates spatial learning and working memory.

Latencies to find the hidden platform were measured daily and theresults of the second daily test (that evaluates working memory) wereaveraged per group. On the fifth day (Day 5) of the Morris water maze(shown in FIG. 5) there was a statistically marginally significantdifference (p<0.075, one tailed t-test) between the non-Tg mice and theTau-tg vehicle treated mice [8.45±2.87 sec; n=7 vs. 31.88±13.54 sec;n=6; respectively, mean±S.E.]

Importantly, the improvement in learning was analyzed using t-tests fordependent samples that compared for each group the latency to find theplatform on the first day and on the fifth day of the MWM (a learningcurve). Significant improvement was found in the Tau-Tg NAT treatedgroup (p=0.039) and for the non-Tg group (p=0.007), suggesting acognitive improvement upon treatment with NAT in the“tauopathy”—afflicted mice (FIG. 6).

NAT Treatment Increased Brain-Body Weight Ratio of the Tau-Tg Mice

All mice were weighed before first drug application and again before thedissection (while still alive). Body weights before and after treatmentwere compared by t-test for repeated measures and no statisticaldifference was found (p=0.98). Whole brain was weighed before the brainsections were separated and no significant statistical differencesbetween the groups were found. However, Brain-Body weight ratio may beused to measure brain mass decrease possibly indicating neuronaldegradation (Bassan et al., (2009) J Matern Fetal Neonatal Med, 1-6).

Brain-Body weight ratio was calculated for each mouse and averaged pergroup [TAU-Tg+NAT 0.0148+0.0009, TAU-Tg+Vh 0.0117+0.0007, w.t. control0.0152+0.0005]. The difference between group averages was confirmed byone way ANOVA that showed a significant difference between the threeexperimental groups with p=0.006. Tukey HSD post-hoc test showed asignificant difference between the NAT and vehicle treated TAU-Tg groups(p=0.030) and between the non-Tg. and the vehicle treated Tau-tg animals(p=0.007). NAT treated Tg mice were not different from the non-Tg group(p=0.909) suggesting that NAT treatment protected the brain fromneurodegeneration (FIG. 7).

Increase in the Relative Amount of Nuclear ADNP in NAT Treated Tau-TgMice

ADNP (Activity-Dependent Neuroprotective Protein) is a protein highlyexpressed in the brain as well as other tissues and shown to be secretedfrom glial cells and further involved in neuroprotection in a variety ofcytotoxic damages. It had been shown that ADNP expression is correlatedwith the need of brain protection (Gozes (2007) Pharmacol Ther, 114,146-154).

In this study, mouse endogenous ADNP levels were quantified byimmunoblotting with ADNP specific antibodies. One way ANOVA analysisshowed a significant difference between the three experimental groups(p=0.0079). Tukey HSD post-hoc test revealed a difference between theNAT and vehicle treated TAU-Tg groups (p=0.0028) and between the vehicletreated TAU-Tg and non-TG group (p=0.0097) (FIG. 8).

Worthy of note, ADNP levels in non-Tg mice are as high as in NAT treatedmice. This high level could be related to the degree of brainprotection. However, actin was used also (FIG. 9) and showed nostatistical difference among the tested groups.

It will be appreciated that this invention describes a new class oftubulin-binding peptide mimetics, including those comprising peptideswith similarity to NAP or SAL for providing neurotrophic andneuroprotective activity and potential additional therapeuticactivities. Modifications include conventional replacements, addition of40 amino acid N- or C-terminal, lipophylization, acetylation etc.

The examples set out above are intended to be exemplary of the effectsof the invention, and are not intended to limit the embodiments or scopeof the invention contemplated by the claims set out below. Othervariants of the invention will be readily apparent to one of ordinaryskill in the art and are encompassed by the appended claims. Allpublications, databases, Genbank sequences, GO terms, patents, andpatent applications cited in this specification are incorporated byreference in their entireties, as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A NAP-like peptide mimetic or SAL-like peptidemimetic, wherein the NAP-like or SAL-like peptide mimetic has theformula (R¹)_(a)—(R²)—(R³)_(b) (SEQ ID NO:X) wherein: R¹ is an aminoacid sequence comprising from 1 to about 40 amino acids wherein eachamino acid is independently selected from the group consisting ofnaturally occurring amino acids and amino acid analogs; R² is a memberselected from the group consisting of (SEQ ID NO: 4) NATLSIHQ,(SEQ ID NO: 6) STPTAIPQ, (SEQ ID NO: 2) NAVLSIHQ, (SEQ ID NO: 3)NATLSVHQ, (SEQ ID NO: 5) NATLSIVHQ, (SEQ ID NO: 7) NTPVSIPQ,(SEQ ID NO: 8) APVSIPQ, (SEQ ID NO: 9) NTPISIPQ, (SEQ ID NO: 10)NAPVSIP, (SEQ ID NO: 11) NAPVAVPQ, (SEQ ID NO: 12) NARVSIPQ,(SEQ ID NO: 13) DAPVSVPQ, (SEQ ID NO: 20) ALLRSIPA, (SEQ ID NO: 21)ALLRSIP, (SEQ ID NO: 22) AMLRSIPA, (SEQ ID NO: 23) ALLRAIPA,(SEQ ID NO: 24) SALLRSIP, (SEQ ID NO: 25) SALLRAIP, (SEQ ID NO: 26)ALLRTIPA, and (SEQ ID NO: 27) ALLRSVPA;

R³ is an amino acid sequence comprising from 1 to about 40 amino acidswherein each amino acid is independently selected from the groupconsisting of naturally occurring amino acids and amino acid analogs;and a and b are independently selected and are equal to zero or one,with the proviso that the NAP-like or SAL-like peptide mimetic does notcomprise the sequence NAPVSIPQ (SEQ ID NO:1) or SALLRSIPA (SEQ IDNO:19).
 2. The NAP-like peptide mimetic or SAL-like peptide mimetic ofclaim 1, wherein R² is a member selected from the group consisting ofNATLSIHQ (SEQ ID NO:4) and STPTAIPQ (SEQ ID NO:6).
 3. The NAP-likepeptide mimetic or SAL-like peptide mimetic of claim 1, wherein a and bare equal to zero.
 4. The NAP-like peptide mimetic or SAL-like peptidemimetic of claim 1, wherein at least one amino acid of R² is a D-aminoacid.
 5. The NAP-like peptide mimetic or SAL-like peptide mimetic ofclaim 1, wherein each amino acid of R² is a D-amino acid.
 6. TheNAP-like peptide mimetic or SAL-like peptide mimetic of claim 1, whereinthe NAP-like peptide mimetic or SAL-like peptide mimetic furthercomprises at least one protecting group.
 7. The NAP-like peptide mimeticor SAL-like peptide mimetic of claim 1, wherein the peptide mimetic isNATLSIHQ (SEQ ID NO:4).
 8. The NAP-like peptide mimetic or SAL-likepeptide mimetic of claim 1, wherein the peptide mimetic is STPTAIPQ (SEQID NO:6).
 9. The NAP-like peptide mimetic or SAL-like peptide mimetic ofclaim 7 or 8, wherein at least one amino acid is a D-amino acid.
 10. TheNAP-like peptide mimetic or SAL-like peptide mimetic of claim 7 or 8,wherein each amino acid is a D-amino acid.
 11. The NAP-like peptidemimetic or SAL-like peptide mimetic of claim 7 or 8, wherein theNAP-like peptide mimetic or SAL-like peptide mimetic further comprisesat least one protecting group.
 12. A pharmaceutical compositioncomprising the NAP-like peptide mimetic or SAL-like peptide mimetic ofclaim
 1. 13. The pharmaceutical composition of claim 12, furthercomprising a neuroprotective polypeptide comprising an amino acidsequence selected from the group consisting of NAPVSIPQ (SEQ ID NO:1)and SALLRSIPA (SEQ ID NO:19).
 14. A method of treating or preventing aneurodegenerative disorder, a cognitive deficit, an autoimmune disorder,peripheral neurotoxicity, motor dysfunction, sensory dysfunction,anxiety, depression, schizophrenia, psychosis, a condition related tofetal alcohol syndrome, a condition involving retinal degeneration, adisorder affecting learning and memory, or a neuropsychiatric disorderin a subject, the method comprising the step of administering atherapeutically effective amount of a NAP-like peptide mimetic orSAL-like peptide mimetic of claim 1, to a subject in need thereof,thereby treating or preventing the neurodegenerative disorder, thecognitive deficit, the autoimmune disorder, peripheral neurotoxicity,motor dysfunction, sensory dysfunction, anxiety, depression,schizophrenia, psychosis, the condition related to fetal alcoholsyndrome, the condition involving retinal degeneration, the disorderaffecting learning and memory, or the neuropsychiatric disorder in thesubject.
 15. The method of claim 14, wherein the NAP-like peptidemimetic is NATLSIHQ (SEQ ID NO:4).
 16. The method of claim 14, whereinthe NAP-like peptide mimetic is STPTAIPQ (SEQ ID NO:6).
 17. The methodof claim 14, wherein the NAP-like peptide mimetic or SAL-like peptidemimetic is administered intranasally.
 18. The method of claim 14,wherein the NAP-like peptide mimetic or SAL-like peptide mimetic isadministered orally.
 19. The method of claim 14, wherein the NAP-likepeptide mimetic or SAL-like peptide mimetic is administeredintravenously or subcutaneously.